Sustained polypeptide expression from synthetic, modified RNAs and uses thereof

ABSTRACT

Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypeptide or altering the developmental potential. Accordingly, provided herein are compositions, methods, and kits comprising synthetic, modified RNAs for changing the phenotype of a cell or cells. These methods, compositions, and kits comprising synthetic, modified RNAs can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims is a continuation application under 35 U.S.C. § 120 of co-pending application U.S. Ser. No. 14/311,545, filed on Jun. 23, 2014, which is a continuation application under 35 U.S.C. § 120 of U.S. Ser. No. 13/088,099, filed on Apr. 15, 2011, now U.S. Pat. No. 8,802,438, issued Jul. 23, 2014, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 61/325,003 filed on Apr. 16, 2010 and U.S. Provisional Patent Application Ser. No. 61/387,220 filed on Sep. 28, 2010, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 31, 2017, is named 67442PCT.txt and is 7,199,441 bytes in size.

FIELD OF THE INVENTION

The field of the invention relates to synthetic, modified RNAs and uses thereof.

BACKGROUND

The ability to change the phenotype of a cell or cells, either to express a desired protein or to change the differentiated phenotype of the cell to that of another, desired cell type, has applications in both research and therapeutic settings. The phenotype of a cell is most commonly modified by expression of protein(s) from exogenous DNA or from recombinant viral vectors. These approaches have the potential for unintended mutagenic effects.

One area of interest is the modification of cellular differentiation such that cells are directed to different developmental lineages. As one example, generating insulin-producing pancreatic β cells from acinar pancreatic cells or other somatic cell types, has the potential to treat diabetes. As but one other example, the ability to redifferentiate a tumor cell or tumor stem cell to a non-cancerous cell type can provide a therapy for cancer. Current protocols for altering cell fate tend to focus on the expression of factors, such as differentiation factors, dedifferentiation factors, transdifferentiation factors, and reprogramming factors, using viral- or DNA-mediated expression.

An area of recent focus is the production of pluripotent or multipotent stem cells from non-embryonic sources. Induction of pluripotency was originally achieved by Yamanaka and colleagues using retroviral vectors to enforce expression of four transcription factors, KLF4, c-MYC, OCT4, and SOX2 (KMOS) (Takahashi, K. and S. Yamanaka, Cell, 2006. 126(4): p. 663-76; Takahashi, K., et al., Cell, 2007. 131(5): p. 861-72). Attempts to derive induced pluripotent stem (iPS) cells have also been made using excisable lentiviral and transposon vectors, or through repeated application of transient plasmid, episomal, and adenovirus vectors (Chang, C.-W., et al., Stem Cells, 2009. 27(5): p. 1042-1049; Kaji, K., et al., Nature, 2009. 458(7239): p. 771-5; Okita, K., et al., Science, 2008. 322(5903): p. 949-53; Stadtfeld, M., et al., Science, 2008. 322(5903): p. 945-9; Woltjen, K., et al., Nature, 2009; Yu, J., et al., Science, 2009: p. 1172482; Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8): p. 348-62). Human pluripotent cells have also been derived using two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim, D., et al., Cell Stem Cell, 2009. 4(6): p. 472-476; Zhou, H., et al., Cell Stem Cell, 2009. 4(5): p. 381-4), and infectious transgene delivery using the Sendai virus, which has a completely RNA-based reproductive cycle (Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8): p. 348-62).

SUMMARY

Provided herein are compositions, methods, and kits for changing the phenotype of a cell or cells. These methods, compositions, and kits can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type. Significantly, the methods, compositions, and kits described herein do not utilize exogenous DNA or viral vector-based methods for the expression of protein(s), and thus, do not cause permanent modification of the genome or have the potential for unintended mutagenic effects.

The compositions, methods, and kits described herein are based upon the direct introduction of synthetic RNAs into a cell, which, when translated, provide a desired protein or proteins. Higher eukaryotic cells have evolved cellular defenses against foreign, “non-self,” RNA that ultimately result in the global inhibition of cellular protein synthesis, resulting in cellular toxicity. This response involves, in part, the production of Type I or Type II interferons, and is generally referred to as the “interferon response” or the “cellular innate immune response.” The cellular defenses normally recognize synthetic RNAs as foreign, and induce this cellular innate immune response. The inventors have recognized that the ability to achieve sustained or repeated expression of an exogenously directed protein using synthetic RNA is hampered by the induction of this innate immune response. In the methods described herein, the effect of the cellular innate immune response is mitigated by using synthetic RNAs that are modified in a manner that avoids or reduces the response. Avoidance or reduction of the innate immune response permit sustained expression from exogenously introduced RNA necessary, for example, to modify the developmental phenotype of a cell. In one aspect, sustained expression is achieved by repeated introduction of synthetic, modified RNAs into a target cell or its progeny.

The modified, synthetic RNAs described herein, in one aspect, can be introduced to a cell in order to induce exogenous expression of a protein of interest in a cell. The ability to direct exogenous expression of a protein of interest using the modified, synthetic RNAs described herein is useful, for example, in the treatment of disorders caused by an endogenous genetic defect in a cell or organism that impairs or prevents the ability of that cell or organism to produce the protein of interest. Accordingly, in some embodiments, compositions and methods comprising the modified, synthetic RNAs described herein can be used for the purposes of gene therapy.

The modified, synthetic RNAs described herein can advantageously be used in the alteration of cellular fates and/or developmental potential. The ability to express a protein from an exogenous RNA permits both the alteration or reversal of the developmental potential of a cell, i.e., the reprogramming of the cell, and the directed differentiation of a cell to a more differentiated phenotype. A critical aspect in altering the developmental potential of a cell is the requirement for sustained and prolonged expression of one or more developmental potential altering factors in the cell or its immediate progeny. Traditionally, such sustained expression has been achieved by introducing DNA or viral vectors to a cell. These traditional approaches have limited therapeutic utility due to the potential for insertional mutagenesis. The compositions and methods described herein completely avoid such risks related to genomic alterations.

One of the areas that can most benefit from the ability to express a desired protein or proteins over a sustained period of time from exogenous synthetic, modified RNAs as described herein is the generation of pluripotent or multipotent cells from cells initially having a more differentiated phenotype. In this aspect, synthetic, modified RNAs encoding a reprogramming factor or factors are used to reprogram cells to a less differentiated phenotype, i.e., having a greater developmental potential. Unexpectedly, the inventors have discovered that the synthetic, modified RNAs described herein permit both dramatically enhanced efficiency and rate of cellular reprogramming relative to DNA- or viral vector-mediated reprogramming methods.

A major goal of stem cell technology is to make the stem cell differentiate into a desired cell type, i.e., directed differentiation. Not only are the compositions and methods described herein useful for reprogramming cells, they are also applicable to this directed differentiation of cells to a desired phenotype. That is, the same technology described herein for reprogramming is directly applicable to the differentiation of the reprogrammed cell, or any other stem cell or precursor cell, for that matter, to a desired cell type.

Accordingly, in one aspect, provided herein are synthetic, modified RNA molecules encoding a polypeptide, where the synthetic, modified RNA molecule comprises one or more modifications, such that introducing the synthetic, modified RNA molecule to a cell results in a reduced innate immune response relative to a cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising the one or more modifications.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects described herein, the innate immune response comprises expression of a Type I or Type II interferon.

In some embodiments of this aspect and all such aspects described herein, the innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.

In another aspect, provided herein is a cell contacted with a synthetic, modified RNA molecule encoding a polypeptide, or a progeny cell of the contacted cell, where the synthetic, modified RNA molecule comprises one or more modifications, such that introducing the synthetic, modified RNA molecule to the cell results in a reduced innate immune response relative to the cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising the one or more modifications.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule contacted with the cell comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule contacted with the cell further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule contacted with the cell does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule contacted with the cell further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule contacted with the cell is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects described herein, the innate immune response comprises expression of a Type I or Type II interferon, and the expression of the Type I or Type II interferon is not increased more than three-fold compared to a reference from a cell which has not been contacted with the synthetic modified RNA molecule.

In some embodiments of this aspect and all such aspects described herein, the innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, and where the expression of the one of more IFN signature genes is not increased more than six-fold compared to a reference from a cell which has not been contacted with the synthetic modified RNA molecule.

In some embodiments of this aspect and all such aspects described herein, the polypeptide encoded by the synthetic, modified RNA molecule introduced to the cell alters a function or a developmental phenotype of the cell. In some such embodiments, the developmental phenotype is a developmental potential. In some embodiments, the developmental potential is decreased. In some embodiments, the developmental potential is increased.

In some embodiments of this aspect and all such aspects described herein, the polypeptide encoded by the synthetic, modified RNA molecule is a reprogramming factor, a differentiation factor, or a de-differentiation factor.

In another aspect, provided herein is a cell contacted with a synthetic, modified RNA molecule encoding a polypeptide, or a progeny cell of the contacted cell, where expression of the encoded polypeptide in the cell alters a function or a developmental phenotype of the cell, and where the synthetic, modified RNA molecule comprises one or more modifications, such that introducing the synthetic, modified RNA molecule to the cell results in a reduced innate immune response relative to the cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising the one or more modifications.

In some embodiments of this aspect and all such aspects described herein, the developmental phenotype altered by expression of the polypeptide encoded by the synthetic, modified RNA molecule is a developmental potential. In some such embodiments of this aspect, the developmental potential is decreased. In other such embodiments of this aspect, the developmental potential is increased.

In some embodiments of these aspects and all such aspects described herein, the polypeptide encoded by the synthetic, modified RNA molecule is a reprogramming factor, a differentiation factor, or a de-differentiation factor.

In another aspect, provided herein is a pluripotent cell, where the pluripotent cell is not an embryonic stem cell, and where the cell was not induced by viral expression of one or more reprogramming factors, and where the cell, when subjected to an unsupervised hierarchical cluster analysis, clusters more closely to an embryonic stem cell than does a pluripotent cell induced by viral expression of one or more reprogramming factors, exogenous protein introduction of one or more reprogramming factors, small molecule mediated expression or induction of one or more reprogramming factors, or any combination thereof.

In one such aspect, provided herein is pluripotent cell, where the pluripotent cell is not an embryonic stem cell, and where the cell was not induced by viral expression of one or more reprogramming factors, and where the cell subjected to an unsupervised hierarchical cluster analysis clusters more closely to a human embryonic stem cell than does a pluripotent cell induced by viral expression of one or more reprogramming factors.

In some embodiments of these aspects and all such aspects described herein, the unsupervised hierarchical cluster analysis is performed on the pluripotent cells using a Euclidean distance with average linkage method, in which the similarity metric for comparison between different cells is indicated on the height of cluster dendrogram.

In some embodiments of these aspects and all such aspects described herein, the unsupervised hierarchical cluster analysis is performed on the pluripotent cells using a data set selected from the group consisting of gene expression data, protein expression data, DNA methylation data, histone modification data, and microRNA data.

In some embodiments of these aspects and all such aspects described herein, the pluripotent cell is generated from a precursor somatic cell contacted with at least one synthetic, modified RNA encoding a reprogramming factor.

In some embodiments of these aspects and all such aspects described herein, the pluripotent cell is generated from a precursor human somatic cell.

Another aspect provides a cell comprising an exogenously introduced modified, synthetic RNA encoding a developmental potential altering factor.

In some embodiments of this aspect and all such aspects described herein, the cell is a human cell. In other embodiments of this aspect and all such aspects described herein, the cell is not a human cell.

In some embodiments of this aspect and all such aspects described herein, the cell or its immediate precursor cell(s) has been subjected to at least 3 separate rounds of contacting with the exogenously introduced modified synthetic RNA encoding the developmental potential altering factor.

In some embodiments of this aspect and all such aspects described herein, the cell has a reduced expression of a Type I or Type II IFN relative to a cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified, synthetic RNA encoding the developmental potential altering factor.

In some embodiments of this aspect and all such aspects described herein, the cell has a reduced expression of at least one IFN-signature gene relative to a cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the developmental potential altering factor.

In one such embodiment of this aspect and all such aspects described herein, the IFN-signature gene is selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.

In some embodiments of this aspect and all such aspects described herein, the developmental potential altering factor is a reprogramming factor, a differentiation factor, or a de-differentiation factor.

In one such embodiment of this aspect and all such aspects described herein, the reprogramming factor is selected from the group consisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some embodiments of this aspect and all such aspects described herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects described herein, the cell or its immediate precursor cell(s) is derived from a somatic cell, a partially reprogrammed somatic cell, a pluripotent cell, a multipotent cell, a differentiated cell, or an embryonic cell.

In another aspect, provided herein is a composition comprising at least one modified, synthetic RNA encoding a reprogramming factor, and cell growth media.

In some embodiments of this aspect and all such aspects described herein, the composition permits an efficiency of pluripotent cell generation from a starting population of somatic cells of at least 1%.

In some embodiments of this aspect and all such aspects described herein, the composition permits a rate of pluripotent cell generation from a starting population of somatic cells of less than 25 days and greater than 7 days.

In one embodiment of this aspect and all such aspects described herein, the reprogramming factor is selected from the group consisting of: OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28. In some embodiments of this aspect and all such aspects described herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects described herein, the composition comprises at least 3 synthetic, modified, RNAs encoding at least 3 different reprogramming factors. In one such embodiment, the at least 3 different reprogramming factors encoded by the at least 3 synthetic, modified RNAs are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, and LIN-28.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor is further treated with an alkaline phosphatase.

Another aspect provides a pluripotent cell generated using any of the compositions described herein.

In one aspect, provided herein is a cell composition comprising a pluripotent cell clone isolated from a population of somatic cells contacted a plurality of times with at least one synthetic, modified RNA encoding a developmental potential altering factor.

In some embodiments of this aspect and all such aspects described herein, the population of somatic cells is a population of human somatic cells.

In some embodiments of this aspect and all such aspects described herein, the pluripotent cell clone subjected to an unsupervised hierarchical cluster analysis clusters more closely to a human embryonic stem cell than does a pluripotent cell clone induced by viral expression of one or more reprogramming factors, exogenous protein introduction of one or more reprogramming factors, small molecule mediated expression or induction of one or more reprogramming factors, or any combination thereof.

Provided herein are methods of altering the developmental potential of a cell. In one aspect, the method comprises contacting with or introducing to a cell population or progeny cells thereof at least one synthetic, modified RNA encoding a developmental potential altering factor. In some embodiments of this aspect and all such aspects described herein, the contacting with or introducing to is performed at least three times.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the developmental potential altering factor is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of determining that the cell population or progeny cells thereof maintain increased viability by measuring viability of the cell population or progeny cells thereof, where the viability of at least 50% of the contacted cell population or progeny cells thereof indicates that the cells maintain increased viability.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of determining that the cell population or progeny cells thereof does not have a significant increase in expression of a Type I or a Type II IFN by measuring expression of a Type I or a Type II IFN in the contacted cell population or progeny cells thereof, where a less than three-fold increase in expression of Type I or Type II IFN in the contacted cell population or progeny cells thereof compared to cells that have not been contacted with the synthetic and modified RNA indicates that the cell population does not have a significant increase in expression of Type I or Type II IFN.

In some such embodiments of this aspect and all such aspects described herein, measuring the expression of Type I or Type II IFN is performed by measuring expression of at least one IFN-signature gene selected from IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, where a less than six-fold increase in expression of the at least one IFN-signature gene compared to the cell population or progeny cells thereof prior to contacting the cell population or progeny cells thereof with the at least one modified and synthetic RNA.

In some embodiments of this aspect and all such aspects described herein, contacting of the cell population or progeny cells thereof is performed in vitro, ex vivo, or in vivo.

Also provided herein are methods for reprogramming a somatic cell into a pluripotent cell. In one aspect, the method comprises contacting a somatic cell population or progeny cells thereof with at least one modified, synthetic RNA encoding at least one reprogramming factor at least five consecutive times.

In some embodiments of this aspect and all such aspects described herein, the at least five consecutive times occur within 25 days.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic, modified RNA encoding the reprogramming factor comprises at least two modified nucleosides. In one such embodiment, the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one such embodiment, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In one such embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the reprogramming factor further comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the reprogramming factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the reprogramming factor further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA molecule encoding the reprogramming factor is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects described herein, the at least one reprogramming factor is selected from: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some embodiments of this aspect and all such aspects described herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects described herein, the at least one reprogramming factor comprises a synthetic and modified RNA encoding OCT4, a synthetic and modified RNA encoding SOX2, a synthetic and modified RNA encoding c-MYC, and a synthetic and modified RNA encoding KLF4. In some embodiments of this aspect and all such aspects described herein, the at least one reprogramming factor further comprises a synthetic and modified RNA molecule encoding LIN28.

In some embodiments of this aspect and all such aspects described herein, a combination of at least three reprogramming selected from the group consisting of a synthetic, modified RNA encoding OCT4, a synthetic, modified RNA encoding SOX2, a synthetic, modified RNA encoding c-MYC, a synthetic, modified RNA encoding KLF4, and a synthetic, modified RNA molecule encoding LIN28, are used in the methods described herein.

In some embodiments of this aspect and all such aspects described herein, the method further comprises determining increased reprogramming efficiency of the somatic cell by measuring efficiency of reprogramming, where efficiency of at least 1% is indicative of increased reprogramming efficiency.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of determining that the somatic cell or progeny cells thereof maintain increased viability by measuring viability of the somatic cell or progeny cells thereof, where viability of at least 50% of the contacted somatic cell or progeny cells thereof indicates that the cells maintain increased viability.

In some embodiments of this aspect and all such aspects described herein, the method further comprises the step of determining that the reprogrammed somatic cell produced by the method has an increased likeness to the potency of an embryonic stem cell by subjecting the pluripotent cell or pluripotent cell population generated by the method to an unsupervised hierarchical cluster analysis and comparing it to a reference from an unsupervised cluster analysis of a pluripotent cell produced by viral expression of one or more of the reprogramming factors, exogenous protein introduction of one or more reprogramming factors, small molecule mediated expression or induction of one or more reprogramming factors, such that if the reprogrammed somatic cell clusters more closely to an embryonic stem cell than it does to a the reference, it has an increased likeness to the potency of embryonic stem cell.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of determining that the reprogrammed somatic cell or progeny cell thereof does not have a significant increase in expression of IFN by measuring expression of at least one IFN-signature gene in the reprogrammed somatic cell or progeny cell thereof, such that if the increase in expression of the at least one IFN-signature gene is less than six-fold compared to a reference from a somatic cell prior to it being subjected to reprogramming indicates that the reprogrammed somatic cell or progeny cell thereof does not have a significant increase in expression of IFN.

In some such embodiments of this aspect and all such aspects described herein, the method further comprises the IFN-signature gene is selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.

In some embodiments of this aspect and all such aspects described herein, the somatic cell population or progeny cells thereof are contacted under a low-oxygen condition.

In some embodiments of this aspect and all such aspects described herein, the method further comprises determining that the reprogrammed somatic cell or progeny thereof expresses sufficient levels of genes to determine pluripotency by measuring expression of at least two genes selected from the group consisting of SOX2, REX1, DNMT3B, TRA-1-60, TRA-1-81, SSEA3, SSEA4, OCT4, and NANOG and comparing the result to a reference from an embryonic stem cell, such that if at least two of the genes are expressed at the level they are expressed in the embryonic stem cell, it indicates that the reprogrammed somatic cell or progeny thereof expresses sufficient levels of genes to determine pluripotency.

In some embodiments of this aspect and all such aspects described herein, contacting of the somatic cell population or progeny cells thereof is performed in vitro, ex vivo, or in vivo.

In some embodiments of this aspect and all such aspects described herein, the somatic cell is a human somatic cell.

Other aspects described herein provide methods of treating subjects in need of cellular therapies. In such aspects, an effective amount of a population of any of the progenitor, multipotent, oligopotent, lineage-restricted, fully or partially differentiated cells, generated using any of the compositions or methods comprising synthetic, modified RNAs described herein, is administered to a subject in need of a cellular therapy.

Accordingly, in one aspect, provided herein is a method of treating a subject in need of a cellular therapy, comprising: administering to a subject in need of a cellular therapy an effective amount of a population of cells having altered developmental potential produced by contacting a cell population or progeny cells thereof with at least one synthetic, modified RNA encoding a developmental potential altering factor for at least three consecutive times.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic and modified RNA encoding a developmental potential altering factor comprises at least two modified nucleosides. In one embodiment of this aspect, the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In one embodiment of this aspect, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic and modified RNA encoding a developmental potential altering factor at least one synthetic, modified RNA further comprises a 5′ cap. In one embodiment of this aspect, the 5′ cap is a 5′ cap analog. In one such embodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic and modified RNA encoding a developmental potential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic, modified RNA encoding a developmental potential altering factor further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof.

In some embodiments of this aspect and all such aspects described herein, the contacting at least three consecutive times are at least 24 hours apart. In some embodiments of this aspect and all such aspects described herein, the contacting at least three consecutive times occur within 15 days.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of obtaining an autologous cell from the subject and generating a population of cells having altered developmental potential from the autologous cell by contacting the cell population or progeny cells thereof with at least one synthetic, modified RNA encoding a developmental potential altering factor for at least three consecutive times.

In some embodiments of this aspect and all such aspects described herein, the method further comprises a step of determining that the population of cells having altered developmental potential does not have a significant increase in expression of Type I or Type II IFN prior to administering the population of cells having altered developmental potential to the subject, the step comprising measuring expression of Type I or Type II IFN, where expression that is less than three-fold compared to a reference from a cell that has not been subject to a treatment to alter developmental potential indicates that the population of cells having altered developmental potential does not have a significant increase in expression of Type I or Type II IFN.

In some such embodiments of this aspect and all such aspects described herein, the expression of Type I or Type II IFN expression is measured by measuring expression of at least one IFN-signature gene selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, and where increase of less than six-fold of the at least two IFN-signature genes indicates that the population of cells having altered developmental potential does not have a significant increase in expression of Type I or Type II IFN.

In some embodiments of this aspect and all such aspects described herein, the altered developmental potential is pluripotency.

In some such embodiments of this aspect and all such aspects described herein, the developmental potential altering factor is a reprogramming factor selected from the group consisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some embodiments of this aspect and all such aspects described herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects described herein, the population of cells having altered developmental potential is of a lineage selected from one of an ecotodermal lineage, a mesodermal lineage, or an endodermal lineage.

In some embodiments of this aspect and all such aspects described herein, the population of cells having altered developmental potential is multipotent. In some embodiments of this aspect and all such aspects described herein, the population of cells having altered developmental potential is oligopotent. In some embodiments of this aspect and all such aspects described herein, the population of cells being administered is partially or fully differentiated.

In some embodiments of this aspect and all such aspects described herein, the population of cells having altered developmental potential is differentiated into at least one differentiated cell population.

Also provided herein are methods for identifying agents that have effects on a cellular phenotype or cellular parameter. In some aspects, provided herein are methods for identifying an agent that has an effect on a cellular phenotype. In one aspect, the method comprises: (a) contacting a cell with a synthetic, modified RNA encoding a polypeptide in an amount and frequency sufficient to alter the phenotype of the cell to that of a desired phenotype; (b) contacting the altered cell with a candidate agent; (c) assaying the desired phenotype in the presence of the candidate agent, where a change in the phenotype in the presence of the candidate agent indicates the agent has an effect on the phenotype.

In some embodiments of this aspect and all such aspects described herein, the polypeptide encoded by the synthetic, modified RNA is a reprogramming factor. In some embodiments of this aspect and all such aspects described herein, the polypeptide encoded by the synthetic, modified RNA is a differentiating factor.

In some embodiments of this aspect and all such aspects described herein, the cell is a pluripotent or multipotent cell.

In some embodiments of this aspect and all such aspects described herein, the cellular phenotype is viability, cell growth, expression of a cell-surface marker, or a functional parameter. In some such embodiments of this aspect and all such aspects described herein, the functional parameter is an electrophysiological parameter, an immunological parameter, or a metabolic parameter. In some embodiments, the metabolic parameter is insulin synthesis or insulin secretion. In some embodiments, the electrophysiological parameter is contractibility.

Also provided herein are kits for altering the phenotype or developmental potential of a cell. In one aspect, provided herein is a kit comprising: a) a container with at least one synthetic, modified RNA molecule comprising at least two modified nucleosides, and b) packaging and instructions therefor.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises a container with cell culture medium.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises an IFN inhibitor. In some embodiments of this aspect and all such aspects described herein, the kit further comprises valproic acid.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic, modified RNA encodes a developmental potential altering factor.

In some embodiments of this aspect and all such aspects described herein, the developmental potential altering factor is a reprogramming factor, a differentiation factor, or a de-differentiation factor.

In some embodiments of this aspect and all such aspects described herein, the synthetic, modified RNA encoding a reprogramming factor in the container has a concentration of 100 ng/μl. In some such embodiments of this aspect and all such aspects described herein, the reprogramming factor is selected from the group consisting of OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some such embodiments of this aspect and all such aspects described herein, the kit comprises at least three of the reprogramming factors. In some such embodiments of this aspect and all such aspects described herein, the at least three reprogramming factors comprise a synthetic, modified RNA encoding OCT4, a synthetic, modified RNA encoding SOX2, a synthetic, modified RNA encoding c-MYC, and a synthetic, modified RNA encoding KLF4. In some such embodiments of this aspect and all such aspects described herein, such that the total concentration of the reprogramming factors in the container is 100 ng/μl, and where OCT4 is provided in molar excess of about three times the concentration of KLF4, SOX-2, and c-MYC. In some such embodiments of this aspect and all such aspects described herein, the kit further comprises a synthetic, modified RNA molecule encoding LIN28.

In some embodiments of this aspect and all such aspects described herein, the kit does not comprise a synthetic, modified RNA encoding c-MYC.

In some embodiments of this aspect and all such aspects described herein, the at least two modified nucleosides of the synthetic, modified RNA are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In some embodiments of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic, modified RNA further comprises a 5′ cap. In some such embodiments of this aspect and all such aspects described herein, the 5′ cap is a 5′ cap analog. In one embodiment of this aspect and all such aspects described herein, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic, modified RNA does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects described herein, the at least one synthetic and modified RNA further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated regions, or any combination thereof. In some such embodiments of this aspect and all such aspects described herein, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof, comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises a non-implantable delivery device or an implantable delivery device to deliver the at least one synthetic, modified RNA. In some such embodiments of this aspect and all such aspects described herein, the non-implantable delivery device is a pen device. In some such embodiments, the implantable delivery device is a pump, semi-permanent stent, or reservoir.

Another aspect provides a kit for reprogramming a somatic cell to an induced pluripotent stem cell, the kit comprising: a) a vial comprising a synthetic, modified RNA encoding an OCT4 reprogramming factor and a buffer; b) a vial comprising a synthetic, modified RNA encoding a SOX2 reprogramming factor and a buffer; c) a vial comprising a synthetic, modified RNA encoding a c-MYC reprogramming factor and a buffer; d) a vial comprising a synthetic, modified RNA encoding a KLF4 reprogramming factor and a buffer; and e) packaging and instructions therefor; where each of the synthetic, modified RNAs encoding a reprogramming factor comprise at least two modified nucleosides.

In some embodiments of this aspect and all such aspects described herein, the at least two modified nucleosides are pseudouridine and 5-methylcytodine.

In some embodiments of this aspect and all such aspects described herein, the concentration in the vial of each of the synthetic, modified RNAs encoding reprogramming factors is 100 ng/μl.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises a vial comprising a synthetic, modified RNA molecule encoding a LIN28 reprogramming factor and a buffer.

In some embodiments of this aspect and all such aspects described herein, the buffer is RNase-free TE buffer at pH 7.0.

In some embodiments of this aspect and all such aspects described herein, the kit further a synthetic, modified RNA encoding a positive control.

In one embodiment of those aspects where a kit is provided to induce reprogramming of a somatic cell to an induced pluripotent stem cell, the kit comprises: a vial comprising a synthetic, modified RNA encoding OCT4 and a buffer; a vial comprising a synthetic, modified RNA encoding SOX2 and a buffer; a vial comprising a synthetic, modified RNA encoding c-MYC and a buffer; a vial comprising a synthetic, modified RNA encoding KLF4 and a buffer; a vial comprising a synthetic, modified RNA molecule encoding LIN28 and a buffer; a vial comprising a synthetic, modified RNA encoding a positive control GFP molecule; and packaging and instructions therefor; where the buffers in each of the vials is RNase-free TE buffer at pH 7.0; and where the synthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 and GFP all comprise pseudouridine and 5-methylcytidine nucleoside modifications. In one embodiment, the concentration of the synthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 and GFP in each of the vials is 100 ng/μl.

Also provided, in another aspect, is a kit for reprogramming a somatic cell to an induced pluripotent stem cell, the kit comprising: a) a container comprising a synthetic, modified RNA encoding an OCT4 reprogramming factor; a synthetic, modified RNA encoding a SOX2 reprogramming factor; a synthetic, modified RNA encoding a c-MYC reprogramming factor; a synthetic, modified RNA encoding a KLF4 reprogramming factor; and a buffer, where each of the synthetic, modified RNAs encoding a reprogramming factor comprises at least two modified nucleosides; and b) packaging and instructions therefor.

In some embodiments of this aspect and all such aspects described herein, the at least two modified nucleosides are pseudouridine and 5-methylcytodine.

In some embodiments of this aspect and all such aspects described herein, the concentration in the container of the synthetic, modified RNAs encoding reprogramming factors is 100 ng/μl.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises a synthetic, modified RNA molecule encoding a LIN28 reprogramming actor.

In some embodiments of this aspect and all such aspects described herein, the kit further comprises a synthetic, modified RNA encoding a positive control.

In some embodiments of this aspect and all such aspects described herein, the buffer is RNase-free TE buffer at pH 7.0.

In some embodiments of this aspect and all such aspects described herein, each of the synthetic, modified RNAs encoding a reprogramming factor further comprise a ligand. In some such embodiments of this aspect and all such aspects described herein, the ligand is a lipid or lipid-based molecule.

Definitions

For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms “developmental potential” or “developmental potency” refer to the total of all developmental cell fates or cell types that can be achieved by a cell upon differentiation. Thus, a cell with greater or higher developmental potential can differentiate into a greater variety of different cell types than a cell having a lower or decreased developmental potential. The developmental potential of a cell can range from the highest developmental potential of a totipotent cell, which, in addition to being able to give rise to all the cells of an organism, can give rise to extra-embryonic tissues; to a “unipotent cell,” which has the capacity to differentiate into only one type of tissue or cell type, but has the property of self-renewal, as described herein; to a “terminally differentiated cell,” which has the lowest developmental potential. A cell with “parental developmental potential” refers to a cell having the developmental potential of the parent cell that gave rise to it.

The term “totipotency” refers to a cell with a developmental potential to make all of the cells in the adult body as well as the extra-embryonic tissues, including the placenta. The fertilized egg (zygote) is totipotent, as are the cells (blastomeres) of the morula (up to the 16-cell stage following fertilization).

The term “pluripotent” as used herein refers to a cell with the developmental potential, under different conditions, to differentiate to cell types characteristic of all three germ cell layers, i.e., endoderm (e.g., gut tissue), mesoderm (including blood, muscle, and vessels), and ectoderm (such as skin and nerve). A pluripotent cell has a lower developmental potential than a totipotent cell. The ability of a cell to differentiate to all three germ layers can be determined using, for example, a nude mouse teratoma formation assay. In some embodiments, pluripotency can also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency of a cell or population of cells generated using the compositions and methods described herein is the demonstration that a cell has the developmental potential to differentiate into cells of each of the three germ layers. In some embodiments, a pluripotent cell is termed an “undifferentiated cell.” Accordingly, the terms “pluripotency” or a “pluripotent state” as used herein refer to the developmental potential of a cell that provides the ability for the cell to differentiate into all three embryonic germ layers (endoderm, mesoderm and ectoderm). Those of skill in the art are aware of the embryonic germ layer or lineage that gives rise to a given cell type. A cell in a pluripotent state typically has the potential to divide in vitro for a long period of time, e.g., greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell” refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers, but not all three. Thus, a multipotent cell can also be termed a “partially differentiated cell.” Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. “Multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc. . . . ), but it cannot form neurons. Accordingly, the term “multipotency” refers to a state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.

The terms “stem cell” or “undifferentiated cell” as used herein, refer to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types, without a specific implied meaning regarding developmental potential (i.e., totipotent, pluripotent, multipotent, etc.). A stem cell is capable of proliferation and giving rise to more such stem cells while maintaining its developmental potential. In theory, self-renewal can occur by either of two major mechanisms. Stem cells can divide asymmetrically, which is known as obligatory asymmetrical differentiation, with one daughter cell retaining the developmental potential of the parent stem cell and the other daughter cell expressing some distinct other specific function, phenotype and/or developmental potential from the parent cell. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. A differentiated cell may derive from a multipotent cell, which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each such stem cell can give rise to, i.e., their developmental potential, can vary considerably. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, known as stochastic differentiation, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Accordingly, the term “stem cell” refers to any subset of cells that have the developmental potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retain the capacity, under certain circumstances, to proliferate without substantially differentiating. In some embodiments, the term stem cell refers generally to a naturally occurring parent cell whose descendants (progeny cells) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. Cells that begin as stem cells might proceed toward a differentiated phenotype, but then can be induced to “reverse” and re-express the stem cell phenotype, a term often referred to as “dedifferentiation” or “reprogramming” or “retrodifferentiation” by persons of ordinary skill in the art.

The term “embryonic stem cell” as used herein refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see, for e.g., U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913; 7,584,479, which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference). Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta, i.e., are not totipotent.

As used herein, the distinguishing characteristics of an embryonic stem cell define an “embryonic stem cell phenotype.” Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell, such that that cell can be distinguished from other cells not having the embryonic stem cell phenotype. Exemplary distinguishing embryonic stem cell phenotype characteristics include, without limitation, expression of specific cell-surface or intracellular markers, including protein and microRNAs, gene expression profiles, methylation profiles, deacetylation profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like. In some embodiments, the determination of whether a cell has an “embryonic stem cell phenotype” is made by comparing one or more characteristics of the cell to one or more characteristics of an embryonic stem cell line cultured within the same laboratory.

The term “somatic stem cell” is used herein to refer to any pluripotent or multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these somatic stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary naturally occurring somatic stem cells include, but are not limited to, neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. In some aspects described herein, a “somatic pluripotent cell” refers to a somatic cell, or a progeny cell of the somatic cell, that has had its developmental potential altered, i.e., increased, to that of a pluripotent state by contacting with, or the introduction of, one or more reprogramming factors using the compositions and methods described herein.

The term “progenitor cell” is used herein to refer to cells that have greater developmental potential, i.e., a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression) relative to a cell which it can give rise to by differentiation. Often, progenitor cells have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct cells having lower developmental potential, i.e., differentiated cell types, or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

As used herein, the term “somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from proliferation of such a cell in vitro. Stated another way, a somatic cell refers to any cell forming the body of an organism, as opposed to a germline cell. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated, pluripotent, embryonic stem cells—is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell,” by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated, the compositions and methods for reprogramming a somatic cell described herein can be performed both in vivo and in vitro (where in vivo is practiced when a somatic cell is present within a subject, and where in vitro is practiced using an isolated somatic cell maintained in culture).

The term “differentiated cell” encompasses any somatic cell that is not, in its native form, pluripotent, as that term is defined herein. Thus, the term a “differentiated cell” also encompasses cells that are partially differentiated, such as multipotent cells, or cells that are stable, non-pluripotent partially reprogrammed, or partially differentiated cells, generated using any of the compositions and methods described herein. In some embodiments, a differentiated cell is a cell that is a stable intermediate cell, such as a non-pluripotent, partially reprogrammed cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. Thus, simply culturing such differentiated or somatic cells does not render these cells non-differentiated cells (e.g. undifferentiated cells) or pluripotent cells. The transition of a differentiated cell (including stable, non-pluripotent partially reprogrammed cell intermediates) to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character upon placement in culture. Reprogrammed and, in some embodiments, partially reprogrammed cells, also have the characteristic of having the capacity to undergo extended passaging without loss of growth potential, relative to parental cells having lower developmental potential, which generally have capacity for only a limited number of divisions in culture. In some embodiments, the term “differentiated cell” also refers to a cell of a more specialized cell type (i.e., decreased developmental potential) derived from a cell of a less specialized cell type (i.e., increased developmental potential) (e.g., from an undifferentiated cell or a reprogrammed cell) where the cell has undergone a cellular differentiation process.

The term “reprogramming” as used herein refers to a process that reverses the developmental potential of a cell or population of cells (e.g., a somatic cell). Stated another way, reprogramming refers to a process of driving a cell to a state with higher developmental potential, i.e., backwards to a less differentiated state. The cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments of the aspects described herein, reprogramming encompasses a complete or partial reversion of the differentiation state, i.e., an increase in the developmental potential of a cell, to that of a cell having a pluripotent state. In some embodiments, reprogramming encompasses driving a somatic cell to a pluripotent state, such that the cell has the developmental potential of an embryonic stem cell, i.e., an embryonic stem cell phenotype. In some embodiments, reprogramming also encompasses a partial reversion of the differentiation state or a partial increase of the developmental potential of a cell, such as a somatic cell or a unipotent cell, to a multipotent state. Reprogramming also encompasses partial reversion of the differentiation state of a cell to a state that renders the cell more susceptible to complete reprogramming to a pluripotent state when subjected to additional manipulations, such as those described herein. Such manipulations can result in endogenous expression of particular genes by the cells, or by the progeny of the cells, the expression of which contributes to or maintains the reprogramming. In certain embodiments, reprogramming of a cell using the synthetic, modified RNAs and methods thereof described herein causes the cell to assume a multipotent state (e.g., is a multipotent cell). In some embodiments, reprogramming of a cell (e.g. a somatic cell) using the synthetic, modified RNAs and methods thereof described herein causes the cell to assume a pluripotent-like state or an embryonic stem cell phenotype. The resulting cells are referred to herein as “reprogrammed cells,” “somatic pluripotent cells,” and “RNA-induced somatic pluripotent cells.” The term “partially reprogrammed somatic cell” as referred to herein refers to a cell which has been reprogrammed from a cell with lower developmental potential by the methods as disclosed herein, such that the partially reprogrammed cell has not been completely reprogrammed to a pluripotent state but rather to a non-pluripotent, stable intermediate state. Such a partially reprogrammed cell can have a developmental potential lower that a pluripotent cell, but higher than a multipotent cell, as those terms are defined herein. A partially reprogrammed cell can, for example, differentiate into one or two of the three germ layers, but cannot differentiate into all three of the germ layers.

The term “developmental potential altering factor,” as used herein, refers to a factor such as a protein or RNA, the expression of which alters the developmental potential of a cell, e.g., a somatic cell, to another developmental state, e.g., a pluripotent state. Such an alteration in the developmental potential can be a decrease (i.e., to a more differentiated developmental state) or an increase (i.e., to a less differentiated developmental state). A developmental potential altering factor, can be for example, an RNA or protein product of a gene encoding a reprogramming factor, such as SOX2, an RNA or protein product of a gene encoding a cell-type specific polypeptide transcription factor, such as myoD, a microRNA, a small molecule, and the like.

The term a “reprogramming factor,” as used herein, refers to a developmental potential altering factor, as that term is defined herein, such as a protein, RNA, or small molecule, the expression of which contributes to the reprogramming of a cell, e.g. a somatic cell, to a less differentiated or undifferentiated state, e.g. to a cell of a pluripotent state or partially pluripotent state. A reprogramming factor can be, for example, transcription factors that can reprogram cells to a pluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC, and the like, including as any gene, protein, RNA or small molecule, that can substitute for one or more of these in a method of reprogramming cells in vitro. In some embodiments, exogenous expression of a reprogramming factor, using the synthetic modified RNAs and methods thereof described herein, induces endogenous expression of one or more reprogramming factors, such that exogenous expression of one or more reprogramming factors is no longer required for stable maintenance of the cell in the reprogrammed or partially reprogrammed state. “Reprogramming to a pluripotent state in vitro” is used herein to refer to in vitro reprogramming methods that do not require and/or do not include nuclear or cytoplasmic transfer or cell fusion, e.g., with oocytes, embryos, germ cells, or pluripotent cells. A reprogramming factor can also be termed a “de-differentiation factor,” which refers to a developmental potential altering factor, as that term is defined herein, such as a protein or RNA, that induces a cell to de-differentiate to a less differentiated phenotype, that is a de-differentiation factor increases the developmental potential of a cell.

As used herein, the term “differentiation factor” refers to a developmental potential altering factor, as that term is defined herein, such as a protein, RNA, or small molecule, that induces a cell to differentiate to a desired cell-type, i.e., a differentiation factor reduces the developmental potential of a cell. In some embodiments, a differentiation factor can be a cell-type specific polypeptide, however this is not required. Differentiation to a specific cell type can require simultaneous and/or successive expression of more than one differentiation factor. In some aspects described herein, the developmental potential of a cell or population of cells is first increased via reprogramming or partial reprogramming using synthetic, modified RNAs, as described herein, and then the cell or progeny cells thereof produced by such reprogramming are induced to undergo differentiation by contacting with, or introducing, one or more synthetic, modified RNAs encoding differentiation factors, such that the cell or progeny cells thereof have decreased developmental potential.

In the context of cell ontogeny, the term “differentiate”, or “differentiating” is a relative term that refers to a developmental process by which a cell has progressed further down a developmental pathway than its immediate precursor cell. Thus in some embodiments, a reprogrammed cell as the term is defined herein, can differentiate to a lineage-restricted precursor cell (such as a mesodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as a tissue specific precursor, for example, a cardiomyocyte precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.

As used herein, the term “cell-type specific polypeptide” refers to a polypeptide that is expressed in a cell having a particular phenotype (e.g., a muscle cell, a pancreatic β cell) but is not generally expressed in other cell types with different phenotypes. As but one example, MyoD is expressed specifically in muscle cells but not in non-muscle cells, thus MyoD is a cell-type specific polypeptide.

As used herein, the term “without the formation of a pluripotent intermediate cell” refers to the transdifferentiation of one cell type to another cell type, preferably, in one step; thus a method that modifies the differentiated phenotype or developmental potential of a cell without the formation of a pluripotent intermediate cell does not require that the cell be first dedifferentiated (or reprogrammed) and then differentiated to another cell type. Instead, the cell type is merely “switched” from one cell type to another without going through a less differentiated phenotype. Accordingly, transdifferentiation refers to a change in the developmental potential of a cell whereby the cell is induced to become a different cell having a similar developmental potential, e.g., a liver cell to a pancreatic cell, a pancreatic a cell into a pancreatic β cell, etc.

The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. In some embodiments, an expression product is transcribed from a sequence that does not encode a polypeptide, such as a microRNA.

As used herein, the term “transcription factor” refers to a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transcription of genetic information from DNA to RNA.

As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

The term “exogenous” as used herein refers to a nucleic acid (e.g., a synthetic, modified RNA encoding a transcription factor), or a protein (e.g., a transcription factor) that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found, or in which it is found in lower amounts. A factor (e.g. a synthetic, modified RNA encoding a transcription factor, or a protein, e.g., a polypeptide) is considered exogenous if it is introduced into an immediate precursor cell or a progeny cell that inherits the substance. In contrast, the term “endogenous” refers to a factor or expression product that is native to the biological system or cell (e.g., endogenous expression of a gene, such as, e.g., SOX2 refers to production of a SOX2 polypeptide by the endogenous gene in a cell). In some embodiments, the introduction of one or more exogenous factors to a cell, e.g., a developmental potential altering factor, using the compositions and methods comprising synthetic, modified RNAs described herein, induces endogenous expression in the cell or progeny cell(s) thereof of a factor or gene product necessary for maintenance of the cell or progeny cell(s) thereof in a new developmental potential.

The term “isolated” or “partially purified” as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered “isolated”.

The term “isolated cell” as used herein refers to a cell that has been removed from an organism in which it was originally found, or a descendant of such a cell. Optionally the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell or population of cells from which it descended) was isolated.

The term “isolated population” with respect to an isolated population of cells as used herein refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a “substantially pure” population of cells as compared to the heterogeneous population from which the cells were isolated or enriched. In some embodiments, the isolated population is an isolated population of pluripotent cells which comprise a substantially pure population of pluripotent cells as compared to a heterogeneous population of somatic cells from which the pluripotent cells were derived.

The term “immediate precursor cell” is used herein to refer to a parental cell from which a daughter cell has arisen by cell division.

As used herein, the terms “synthetic, modified RNA” or “modified RNA” refer to an RNA molecule produced in vitro, which comprise at least one modified nucleoside as that term is defined herein below. The synthetic, modified RNA composition does not encompass mRNAs that are isolated from natural sources such as cells, tissue, organs etc., having those modifications, but rather only synthetic, modified RNAs that are synthesized using in vitro techniques. The term “composition,” as applied to the terms “synthetic, modified RNA” or “modified RNA,” encompasses a plurality of different synthetic, modified RNA molecules (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 90, at least 100 synthetic, modified RNA molecules or more). In some embodiments, a synthetic, modified RNA composition can further comprise other agents (e.g., an inhibitor of interferon expression or activity, a transfection reagent, etc.). Such a plurality can include synthetic, modified RNA of different sequences (e.g., coding for different polypeptides), synthetic, modified RNAs of the same sequence with differing modifications, or any combination thereof.

As used herein the term “modified nucleoside” refers to a ribonucleoside that encompasses modification(s) relative to the standard guanine (G), adenine (A), cytidine (C), and uridine (U) nucleosides. Such modifications can include, for example, modifications normally introduced post-transcriptionally to mammalian cell mRNA, and artificial chemical modifications, as known to one of skill in the art.

As used herein, the term “polypeptide” refers to a polymer of amino acids comprising at least 2 amino acids (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 amino acids or more). The terms “protein” and “polypeptide” are used interchangeably herein. As used herein, the term “peptide” refers to a relatively short polypeptide, typically between about 2 and 60 amino acids in length.

As used herein, the term “added co-transcriptionally” refers to the addition of a feature, e.g., a 5′ diguanosine cap or other modified nucleoside, to a synthetic, modified RNA during transcription of the RNA molecule (i.e., the modified RNA is not fully transcribed prior to the addition of the 5′ cap).

The term “contacting” or “contact” as used herein in connection with contacting a cell with one or more synthetic, modified RNAs as described herein, includes subjecting a cell to a culture medium which comprises one or more synthetic, modified RNAs at least one time, or a pluarlity of times, or to a method whereby such a synthetic, modified RNA is forced to contact a cell at least one time, or a plurality of times, i.e., a transfection system. Where such a cell is in vivo, contacting the cell with a synthetic, modified RNA includes administering the synthetic, modified RNA in a composition, such as a pharmaceutical composition, to a subject via an appropriate administration route, such that the compound contacts the cell in vivo.

The term “transfection” as used herein refers the use of methods, such as chemical methods, to introduce exogenous nucleic acids, such as the synthetic, modified RNAs described herein, into a cell, preferably a eukaryotic cell. As used herein, the term transfection does not encompass viral-based methods of introducing exogenous nucleic acids into a cell. Methods of transfection include physical treatments (electroporation, nanoparticles, magnetofection), and chemical-based transfection methods. Chemical-based transfection methods include, but are not limited to, cyclodextrin, polymers, liposomes, and nanoparticles. In some embodiments, cationic lipids or mixtures thereof can be used to transfect the synthetic, modified RNAs described herein, into a cell, such as DOPA, Lipofectamine and UptiFectin. In some embodiments, cationic polymers such as DEAE-dextran or polyethylenimine, can be used to transfect a synthetic, modified RNAs described herein.

The term “transduction” as used herein refers to the use of viral particles or viruses to introduce exogenous nucleic acids into a cell.

As used herein, the term “transfection reagent” refers to any agent that induces uptake of a synthetic, modified RNA into a host cell. Also encompassed are agents that enhance uptake e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 500-fold, at least 100-fold, at least 1000-fold, or more, compared to a synthetic, modified RNA administered in the absence of such a reagent. In one embodiment, a cationic or non-cationic lipid molecule useful for preparing a composition or for co-administration with a synthetic, modified RNA is used as a transfection reagent. In other embodiments, the synthetic, modified RNA comprises a chemical linkage to attach e.g., a ligand, a peptide group, a lipophillic group, a targeting moiety etc. In other embodiments, the transfection reagent comprises a charged lipid, an emulsion, a liposome, a cationic or non-cationic lipid, an anionic lipid, or a penetration enhancer as known in the art or described herein.

As used herein, the term “repeated transfections” refers to repeated transfection of the same cell culture with a synthetic, modified RNA a plurality of times (e.g., more than once or at least twice). In some embodiments, the cell culture is transfected at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more. The transfections can be repeated until a desired phenotype of the cell is achieved.

The time between each repeated transfection is referred to herein as the “frequency of transfection.” In some embodiments, the frequency of transfection occurs every 6 h, every 12 h, every 24 h, every 36 h, every 48 h, every 60 h, every 72 h, every 96 h, every 108 h, every 5 days, every 7 days, every 10 days, every 14 days, every 3 weeks, or more during a given time period in any developmental potential altering regimen, such as a reprogramming, transdifferentiation or differentiation regimen. The frequency can also vary, such that the interval between each dose is different (e.g., first interval 36 h, second interval 48 h, third interval 72 h etc). It should be understood depending upon the schedule and duration of repeated transfections, it will often be necessary to split or passage cells or change or replace the media during the transfection regimen to prevent overgrowth and replace nutrients. For the purposes of the methods described herein, transfections of a culture resulting from passaging an earlier transfected culture is considered “repeated transfection,” “repeated contacting” or “contacting a plurality of times,” unless specifically indicated otherwise.

As used herein, the term “permits repeated transfections” refers to a synthetic, modified RNA or synthetic, modified RNA composition that can be transfected into a given cell culture with reduced cytotoxicity compared to an RNA or RNA composition having the same sequence(s) which lacks modifications to the RNA. As used herein, the term “reduced cytotoxicity” refers to the death of less than 50% of the cells in a cell culture repeatedly transfected with a synthetic, modified RNA or synthetic, modified RNA composition, e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1% or fewer compared to transfection with a composition having the same sequence(s) but lacking modifications to the RNA. The amount of cell death in a culture can be determined using a standard Trypan Blue Exclusion assay, which turns dead cells blue while leaving living cells uncolored. Alternatively “reduced cytotoxicity” can be assessed by measuring apoptosis using e.g., a TUNEL assay. Other useful measures for determining “reduced cytotoxicity” include e.g., flow cytometric and bead based measurements of viability, cell growth, cellularity (measured e.g., microscopically and quantitated by a hemocytometer), global protein production, secretion of cytokines (e.g., Type 1 IFNs), and expression level of interferon response signature genes (e.g., IFIT1, IFITM1, OAS1, IFNA1, IFNB1, PKR, RIG-I, TLR7, TLR8 etc).

As used herein, the term “targeting moiety” refers to an agent that homes to or preferentially associates or binds to a particular tissue, cell type, receptor, infecting agent or other area of interest. The addition of a targeting moiety to an RNA delivery composition will enhance the delivery of the composition to a desired cell type or location. The addition to, or expression of, a targeting moiety in a cell enhances the localization of that cell to a desired location within an animal or subject.

As used herein, the terms “innate immune response” or “interferon response” refers to a cellular defense response initiated by a cell in response to recognition of infection by a foreign organism, such as a virus or bacteria or a product of such an organism, e.g., an RNA lacking the modifications characteristic of RNAs produced in the subject cell. The innate immune response protects against viral and bacterial infection by inducing the death of cells that detect exogenous nucleic acids e.g., by detection of single- or double-stranded RNA that are recognized by pattern recognition receptors such as RIG-I, protein kinase R (PKR), MDA5, or nucleic acid-recognizing Toll-like receptors, e.g., TLR3, TLR7, TLR8, and TLR9, and activating an interferon response. As used herein, the innate immune response or interferon response operates at the single cell level causing cytokine expression, cytokine release, global inhibition of protein synthesis, global destruction of cellular RNA, upregulation of major histocompatbility molecules, and/or induction of apoptotic death, induction of gene transcription of genes involved in apoptosis, anti-growth, and innate and adaptive immune cell activation. Some of the genes induced by type I IFNs include PKR, ADAR (adenosine deaminase acting on RNA), OAS (2′,5′-oligoadenylate synthetase), RNase L, and Mx proteins. PKR and ADAR lead to inhibition of translation initiation and RNA editing, respectively. OAS is a dsRNA-dependent synthetase that activates the endoribonuclease RNase L to degrade ssRNA.

Accordingly, as used herein, the phrases “innate immune response signature” or “interferon response signature” genes refer to the set of genes that are expressed or up-regulated upon an interferon response of a cell, and include, but are not limited to, IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, CDC20, TLR3, TLR7, TLR8, and TLR9.

As used herein, the term “inhibitor of interferon expression or activity” refers to an agent (e.g., small molecule, antibody, antibody fragment, soluble receptor, RNA interference molecule etc.) that: (a) inhibits translation of an interferon polypeptide from an mRNA transcript, (b) inactivates an interferon polypeptide, (c) prevents interferon binding to its receptor or (d) binds/sequesters an interferon polypeptide e.g., for degradation.

As used herein, the term “unsupervised clustering analysis” or “unsupervised cluster analysis” refers to methods used in multivariate analysis to divide up objects into similar groups, or, in some embodiments, groups whose members are all close to one another on various dimensions being measured in the various objects. In cluster analysis, one does not start with any a priori notion of group characteristics. As used herein, “hierarchical cluster analysis” or “hierarchical clustering” refer to a general approach to unsupervised cluster analysis, in which the purpose is to group together objects or records that are “close” to one another. A key component of the analysis is repeated calculation of distance measures between objects, and between clusters once objects begin to be grouped into clusters. The outcome is typically represented graphically as a dendrogram. Hierarchical cluster analysis can be performed using any of a variety of unbiased computational methods, algorithms and software programs known to one of skill in the art that identify clusters or natural data structures from large data sets, such as, for example, gene expression data sets. Such methods include, but are not limited to, bottom-up hierarchical clustering, K-means clustering Affinity Propagation, non-Negative Matrix Factorization, spectral clustering, Self-Organizing Map (SOM) algorithms, and the like. In some embodiments of the aspects described herein, a SOM-based method for use in unsupervised hierarchical clustering analysis of cells contacted with the synthetic, modified RNAs described herein is the Automatic clustering using density-equalized SOM Ensembles (AUTOsome) method as described in A. M. Newman and J. B. Cooper (2010, Cell Stem Cell, 7:258-262) and A. M. Newman and J. B. Cooper (2010, BMC Bioinformatics 2010, 11:117), the contents of each of which are herein incorporated in their entireties by reference. After a clustering analysis of a given data set, such as a gene expression data set, appropriate class-based statistical tests like Student's t-test, ANOVA, or Gene Set Enrichment Analysis can be used to evaluate significance.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

This patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

FIG. 1 depicts a synthetic, modified RNA production flowchart. To construct a template for RNA transcription reactions, the ORF of a gene of interest is first PCR amplified from a cDNA. Long oligonucleotides containing UTR sequences are then joined to the top strand of ORF amplicons by a thermostable DNA ligase, mediated by annealing to splint oligos which bring the desired single-stranded DNA (ssDNA) ends together. An upstream T7 promoter is incorporated in the 5′ UTR fragment. The ssDNA product is amplified using generic primers and TA cloned. A polyA tail is added with a PCR reaction using a T₁₂₀-heeled reverse primer, and the amplicons are used to template IVT reactions. Modified and unmodified nucleobases are used in the IVT reaction. An anti-reverse diguanosine cap analog (ARCA) is included in the IVT reaction at four-fold higher concentration than guanosine triphosphate (GTP), as a result of which an estimated 80% of the product is capped. Spin-column purified IVT product is DNase-treated to eliminate the DNA template. Treatment with a phosphatase is used to remove immunonogenic 5′ triphosphate moieties from the uncapped RNA fraction. The completed synthetic, modified RNA is then re-purified for use in transfections.

FIGS. 2A-2R demonstrate that synthetic, modified RNA overcomes cellular anti-viral responses and can be used to direct and alter cell fate and developmental potential. FIGS. 2A-2D show microscopy images showing keratinocytes transfected 24 hours earlier with 400 ng/well of synthetic, unmodified (No Mods) (FIG. 2A), 5-methyl-cytosine modified (5mC) (FIG. 2B), pseudouridine modified (Psi) (FIG. 2C), or 5mC+Psi modified RNA encoding GFP (FIG. 2D). FIG. 2E shows percent viability and FIG. 2L depicts mean fluorescence intensity of the cells shown in FIGS. 2A-2D as measured by flow cytometry. FIGS. 2F-2K demonstrate quantitative RT-PCR data showing expression of six interferon-regulated genes in BJ fibroblasts 24 hours after transfection with unmodified (No Mods), or synthetic, modified (5mC+Psi) RNA encoding GFP (1200 ng/well), and vehicle and untransfected controls. FIG. 2M depicts flow cytometry histograms showing GFP expression in keratinocytes transfected with 0-160 ng of modified RNA, 24 hours post transfection. FIG. 2N shows microscopy images of keratinocytes co-transfected with synthetic, modified RNAs encoding GFP with a nuclear localization signal, and cytosolic mCherry proteins. FIG. 2O shows growth kinetics of BJ fibroblasts transfected daily with unmodified, or synthetic, modified RNAs encoding a destabilized nuclear-localized GFP, and vehicle and untransfected controls for 10 days. FIG. 2P shows immunostaining for the muscle-specific proteins myogenin and myosin heavy chain (MyHC) in murine C3H/10T1/2 cell cultures 3 days after 3 consecutive daily transfections with a synthetic, modified RNA encoding MYOD. FIGS. 2Q-2R demonstrate sustained GFP expression of synthetic, modified RNA transfected cells described in FIG. 2O at day 10 of transfection shown by fluorescence imaging with bright field overlay (FIG. 2Q), and flow cytometry (FIG. 2R). Error bars indicate s.d., n=3 for all panels.

FIGS. 3A-3F demonstrate penetrant and sustained protein expression mediated by synthetic, modified RNA transfection in diverse human cell types, and effects on cell viability and global gene expression. FIG. 3A depicts analysis of representative flow cytometry data showing penetrance of GFP expression 24-hour post-transfection of six human cell types transfected with 1000 ng of synthetic, modified RNA encoding GFP. Cell types included: human epidermal keratinocytes (HEKs), adipose-derived stem cells (ADSCs), and four different human fibroblast types (BJ, Detroit 551, MRC-5 and dH1f). Error bars show s.d. for triplicate wells. FIGS. 3B and 3D show representative expression time courses for cells transfected with synthetic, modified RNAs encoding high- and low-stability GFP variants (eGFP and d2eGFP, respectively), assayed by flow cytometry. FIG. 3C shows Annexin V staining at indicated days of BJ fibroblasts transfected daily over the course of 10 days. FIG. 3E depicts heatmap data from microarray analysis of BJ fibroblasts transfected for 10 consecutive days with synthetic, modified RNA encoding GFP, vehicle, or untransfected controls. A number of cell stress pathways are shown demonstrating that prolonged transfection with synthetic, modified-RNA does not significantly impact the molecular profile of transfected cells beyond upregulation of a limited number of interferon/NFκB genes highlighted in FIG. 3F. FIG. 3F depicts all genes upregulated greater than 2-fold in synthetic, modified RNA transfected cells versus untransfected cells (right) or vehicle transfected (left) showing induction of number of interferon/NFκB signaling genes consistent with the near but not absolute attenuation of interferon response shown in FIG. 2D.

FIGS. 4A-4F demonstrate generation of RNA-induced pluripotent stem cells (RiPS) using the synthetic, modified RNAs described herein. FIG. 4A shows immunostaining for human KLF4, OCT4, and SOX2 proteins in keratinocytes 15 hours post-transfection with synthetic, modified RNA encoding KLF4, OCT4, or SOX2. FIGS. 4B-4D depicts a time course analysis showing kinetics and stability of KLF4, OCT4, and SOX2 proteins after synthetic, modified RNA transfection, as assayed by flow cytometry following intracellular staining of reach protein. FIG. 4E shows brightfield images taken during the derivation of RNA-iPS cells (RiPS) from dH1f fibroblasts showing early epitheliod morphology (day 6), small hES-like colonies (day 17), and appearance of mature iPS clones after mechanical picking and expansion (day 24). FIG. 4F depicts immunohistochemistry data showing expression of a panel of pluripotency markers in expanded RiPS clones derived from dH1f fibroblasts, Detroit 551 (D551) and MRC-5 fetal fibroblasts, BJ post-natal fibroblasts, and cells derived from a skin biopsy taken from an adult cystic fibrosis patient (CF), shown also in high magnification. BG01 hES cells and BJ1 fibroblasts are included as positive and negative controls, respectively.

FIGS. 5A-5C demonstrate iPS-derivation from five human cell types. FIGS. 5A-5B show an expression time course of low-stability nuclear GFP after a single transfection into keratinocytes, assessed by flow cytometry. Bright-field and GFP images taken at four different time points during a reprogramming experiment are shown. RNA-encoding the low-stability GFP analyzed in the left panel was spiked into the reprogramming cocktail (KMOSL) to visualize sustained protein expression from transfected synthetic, modified RNAs during iPS reprogramming (bottom panel, FIG. 5B). FIG. 5C shows antibody stains of independent RiPS clones derived from cells taken from an adult cystic fibrosis patient (CF cells), BJ postnatal fibroblasts, MRC-5 and Detroit 551 fetal fibroblasts, and human ES-derived dH1f fibroblasts. FIG. 5C panels show cell-surface staining for SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and intracellular staining for OCT4 and NANOG. Control stains of BG01 hES cells, dH1f and BJ fibroblasts are shown. Additional control stains show the specificity of the secondary antibody used for the OCT4 and NANOG intracellular stains.

FIGS. 6A-6B demonstrate efficient RiPS derivation from BJ fibroblasts without passaging. FIG. 6A depicts immunohistochemistry showing expression of pluripotency markers SSEA-4 and TRA-1-60 in a BJ fibroblast reprogramming experiment transfected for 16 days with 600 ng per day of a KMOSL modified RNA cocktail containing a destabilized GFP spike-in. Cultures were fixed for staining at day 18. 50,000 BJ cells were originally seeded onto feeder cells and went unpassaged throughout the course of the experiment. FIG. 6B shows quantification of TRA-1-60 colony count relative to the number of cells seeded.

FIGS. 7A-7I demonstrate a molecular characterization of RiPS cells. FIG. 7A depicts a heatmap showing results of qRT-PCR analysis measuring the expression of pluripotency-associated genes in RiPS cell lines, parental fibroblasts and viral-derived iPS cells relative to hES cell controls. FIG. 7B depicts a heatmap showing results of OCT4 promoter methylation analysis of RiPS cell lines, parental fibroblasts, and hES cell controls. FIGS. 7C-7H demonstrate global gene expression profiles of BJ-, MRC5- and dH1F-derived RiPS cells shown in scatter plots against parental fibroblasts and hES cells with pluripotency-associated transcripts indicated. FIG. 7I depicts a dendrogram showing unsupervised hierarchical clustering of the global expression profiles for RiPS cells, parental fibroblasts, hES cells, and virus-derived iPS cells. The similarity metric for comparison between different cell lines is indicated on the height of cluster dendrogram. One of skill in the art can use these methods to determine the similarity between a RiPS cell and a human embryonic stem cell, or to determine differences between a RiPS cell and a iPS cell made by another method. This figure indicates that a RiPS cell has a higher degree of similarity to an embryonic stem cell than iPS cells derived using retroviruses, i.e., a RiPS cell has an “embryonic stem cell phenotype.”

FIGS. 8A-8C demonstrate trilineage differentiation of RiPS cells. FIG. 8A shows yield and typology of blood-lineage colonies produced by directed differentiation of embryoid bodies in methylcellulose assays with RiPS clones derived from BJ, CF, D551 and MCR5 fibroblasts, and a human ES (H1) control. FIG. 8B depicts immunostaining showing expression of the lineage markers Tuj1 (neuronal, ectodermal), and alpha-fetoprotein (epithelial, endodermal) in RiPS clones from 3 independent RiPS derivations subjected to directed differentiation. FIG. 8C shows hematoxylin and eosin staining of BJ- and dH1F-RiPS-derived teratomas showing histological overview, ectoderm (pigmented epithelia (BJ), neural rosettes (dH1F)), mesoderm (cartilage and muscle, both), and endoderm (gut-like endothelium, both). For blood formation and methylcellulose assays, n=3 for each clone.

FIG. 9 demonstrates teratoma formation and trilineage differentiation of synthetic, modified RNA derived iPS clones in vivo.

FIGS. 10A-10G demonstrates high and surprising efficiency of pluripotency induction by synthetic, modified RNAs. FIG. 10A shows TRA-1-60 horseradish peroxidase (HRP) staining conducted at day 18 of a BJ-RiPS derivation with modified RNAs encoding KMOSL and FIG. 10B shows frequency of TRA-1-60-positive colonies produced in the experiment relative to number of cells initially seeded. Error bars show s.d., n=6 for each condition. FIG. 10C shows TRA-181 HRP, TRA-160 immunofluorescence and Hoechst staining, and FIG. 10D shows colony frequencies for dH1f-RiPS experiments done using 4-factor (KMOS) and 5-factor (KMOSL) synthetic, modified RNA cocktails under 5% O2 or ambient oxygen culture conditions quantified at day 18. Control wells were transfected with equal doses of synthetic, modified RNA encoding GFP. FIGS. 10E-10G compare kinetics and efficiency of retroviral and synthetic, modified RNA reprogramming. Timeline of colony formation is shown in FIG. 10E. TRA-1-60 HRP immuno-staining is shown in FIG. 10F. TRA-1-60 positive colony counts are shown in FIG. 10G of dHlf cells reprogrammed using KMOS retroviruses (MOI=5 of each) or synthetic, modified RNA KMOS cocktails (n=3 for each condition).

FIGS. 11A-11C demonstrate efficient directed differentiation of RiPS cells to terminally differentiated myogenic fate using synthetic, modified RNA. FIG. 11A shows a schematic of experimental design. FIG. 11B shows bright-field and immunostained images showing large, multi-nucleated, myosin heavy chain (MyHC) and myogenin positive myotubes in cells fixed three days after cessation of MYOD synthetic, modified RNA transfection. Synthetic, modified RNA encoding GFP was administered to the controls. FIG. 11C shows a penetrance of myogenic conversion relative to daily RNA dose. Black bars refer to an experiment in which cultures were plated at 10⁴ cells/cm², grey bars to cultures plated at 5×10³ cells/cm². Error bars show s.d. for triplicate wells.

DETAILED DESCRIPTION

Described herein are novel compositions, methods, and kits for changing the phenotype of a cell or cells. These methods, compositions, and kits can be used either to express a desired protein in a cell or tissue, or to change the developmental potential or differentiated phenotype of a cell to that of another, desired cell type. Significantly, the methods and compositions described herein do not utilize exogenous DNA or viral vector-based methods for the expression of protein(s), and thus, do not cause permanent modification of the genome or unintended mutagenic effects.

RNAs and RNA Modification

Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypeptide or altering the developmental potential. As used herein, the term “synthetic, modified RNA” refers to a nucleic acid molecule encoding a factor, such as a polypeptide, to be expressed in a host cell, which comprises at least one modified nucleoside and has at least the following characteristics as the term is used herein: (i) it can be generated by in vitro transcription and is not isolated from a cell; (ii) it is translatable in a mammalian (and preferably human) cell; and (iii) it does not provoke or provokes a significantly reduced innate immune response or interferon response in a cell to which it is introduced or contacted relative to a synthetic, non-modified RNA of the same sequence. A synthetic, modified RNA as described herein permits repeated transfections in a target cell; that is, a cell or cell population transfected with a synthetic, modified RNA molecule as described herein tolerates repeated transfection with such synthetic, modified RNA without significant induction of an innate immune response or interferon response. These three primary criteria for a synthetic, modified RNA molecule described above are described in greater detail below.

First, the synthetic, modified RNA must be able to be generated by in vitro transcription of a DNA template. Methods for generating templates are well known to those of skill in the art using standard molecular cloning techniques. An additional approach to the assembly of DNA templates that does not rely upon the presence of restriction endonuclease cleavage sites is also described herein (termed “splint-mediated ligation”). The transcribed, synthetic, modified RNA polymer can be modified further post-transcription, e.g., by adding a cap or other functional group.

To be suitable for in vitro transcription, the modified nucleoside(s) must be recognized as substrates by at least one RNA polymerase enzyme. Generally, RNA polymerase enzymes can tolerate a range of nucleoside base modifications, at least in part because the naturally occurring G, A, U, and C nucleoside bases differ from each other quite significantly. Thus, the structure of a modified nucleoside base for use in generating the synthetic, modified RNAs described herein can generally vary more than the sugar-phosphate moieties of the modified nucleoside. That said, ribose and phosphate-modified nucleosides or nucleoside analogs are known in the art that permit transcription by RNA polymerases. In some embodiments of the aspects described herein, the RNA polymerase is a phage RNA polymerase. The modified nucleotides pseudouridine, m5U, s2U, m6A, and m5C are known to be compatible with transcription using phage RNA polymerases, while N1-methylguanosine, N1-methyladenosine, N7-methylguanosine, 2′-)-methyluridine, and 2′-O-methylcytidine are not. Polymerases that accept modified nucleosides are known to those of skill in the art.

It is also contemplated that modified polymerases can be used to generate synthetic, modified RNAs, as described herein. Thus, for example, a polymerase that tolerates or accepts a particular modified nucleoside as a substrate can be used to generate a synthetic, modified RNA including that modified nucleoside.

Second, the synthetic, modified RNA must be translatable by the translation machinery of a eukaryotic, preferably mammalian, and more preferably, human cell. Translation generally requires at least a ribosome binding site, a methionine start codon, and an open reading frame encoding a polypeptide. Preferably, the synthetic, modified RNA also comprises a 5′ cap, a stop codon, a Kozak sequence, and a polyA tail. In addition, mRNAs in a eukaryotic cell are regulated by degradation, thus a synthetic, modified RNA as described herein can be further modified to extend its half-life in the cell by incorporating modifications to reduce the rate of RNA degradation (e.g., by increasing serum stability of a synthetic, modified RNA).

Nucleoside modifications can interfere with translation. To the extent that a given modification interferes with translation, those modifications are not encompassed by the synthetic, modified RNA as described herein. One can test a synthetic, modified RNA for its ability to undergo translation and translation efficiency using an in vitro translation assay (e.g., a rabbit reticulocyte lysate assay, a reporter activity assay, or measurement of a radioactive label in the translated protein) and detecting the amount of the polypeptide produced using SDS-PAGE, Western blot, or immunochemistry assays etc. The translation of a synthetic, modified RNA comprising a candidate modification is compared to the translation of an RNA lacking the candidate modification, such that if the translation of the synthetic, modified RNA having the candidate modification remains the same or is increased then the candidate modification is contemplated for use with the compositions and methods described herein. It is noted that fluoro-modified nucleosides are generally not translatable and can be used herein as a negative control for an in vitro translation assay.

Third, the synthetic, modified RNA provokes a reduced (or absent) innate immune response or interferon response by the transfected cell or population of cells thereof. mRNA produced in eukaryotic cells, e.g., mammalian or human cells, is heavily modified, the modifications permitting the cell to detect RNA not produced by that cell. The cell responds by shutting down translation or otherwise initiating an innate immune or interferon response. Thus, to the extent that an exogenously added RNA can be modified to mimic the modifications occurring in the endogenous RNAs produced by a target cell, the exogenous RNA can avoid at least part of the target cell's defense against foreign nucleic acids. Thus, in some embodiments, synthetic, modified RNAs as described herein include in vitro transcribed RNAs including modifications as found in eukaryotic/mammalian/human RNA in vivo. Other modifications that mimic such naturally occurring modifications can also be helpful in producing a synthetic, modified RNA molecule that will be tolerated by a cell. With this as a background or threshold understanding for the requirements of a synthetic, modified RNA, the various modifications contemplated or useful in the synthetic, modified RNAs described herein are discussed further herein below.

RNA Modifications

In some aspects, provided herein are synthetic, modified RNA molecules encoding polypeptides, where the synthetic, modified RNA molecules comprise one or more modifications, such that introducing the synthetic, modified RNA molecules to a cell results in a reduced innate immune response relative to a cell contacted with synthetic RNA molecules encoding the polypeptides not comprising the one or more modifications.

The synthetic, modified RNAs described herein include modifications to prevent rapid degradation by endo- and exo-nucleases and to avoid or reduce the cell's innate immune or interferon response to the RNA. Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. To the extent that such modifications interfere with translation (i.e., results in a reduction of 50% or more in translation relative to the lack of the modification—e.g., in a rabbit reticulocyte in vitro translation assay), the modification is not suitable for the methods and compositions described herein. Specific examples of synthetic, modified RNA compositions useful with the methods described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages. Synthetic, modified RNAs having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage. In other embodiments, the synthetic, modified RNA has a phosphorus atom in its internucleoside linkage(s).

Non-limiting examples of modified internucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is herein incorporated by reference in its entirety.

Modified internucleoside linkages that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of modified oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety.

Some embodiments of the synthetic, modified RNAs described herein include nucleic acids with phosphorothioate internucleoside linkages and oligonucleosides with heteroatom internucleoside linkage, and in particular —CH2-NH—CH2-, —CH2-N(CH3)-O—CH2- [known as a methylene (methylimino) or MMI], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2- [wherein the native phosphodiester internucleoside linkage is represented as —O—P—O—CH2-] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240, both of which are herein incorporated by reference in their entirety. In some embodiments, the nucleic acid sequences featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506, herein incorporated by reference in its entirety.

Synthetic, modified RNAs described herein can also contain one or more substituted sugar moieties. The nucleic acids featured herein can include one of the following at the 2′ position: H (deoxyribose); OH (ribose); F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments, synthetic, modified RNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of a synthetic, modified RNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′ methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2-O—CH2-N(CH2)2.

Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the nucleic acid sequence, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked nucleotides and the 5′ position of 5′ terminal nucleotide. A synthetic, modified RNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

As non-limiting examples, synthetic, modified RNAs described herein can include at least one modified nucleoside including a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof.

In some embodiments of this aspect and all other such aspects described herein, the at least one modified nucleoside is selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′ deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m6₂Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I).

Alternatively, a synthetic, modified RNA can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the oligonucleotide. At a minimum, a synthetic, modified RNA molecule comprising at least one modified nucleoside comprises a single nucleoside with a modification as described herein. It is not necessary for all positions in a given synthetic, modified RNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single synthetic, modified RNA or even at a single nucleoside within a synthetic, modified RNA. However, it is preferred, but not absolutely necessary, that each occurrence of a given nucleoside in a molecule is modified (e.g., each cytosine is a modified cytosine e.g., 5mC). However, it is also contemplated that different occurrences of the same nucleoside can be modified in a different way in a given synthetic, modified RNA molecule (e.g., some cytosines modified as 5mC, others modified as 2′-O-methylcytidine or other cytosine analog). The modifications need not be the same for each of a plurality of modified nucleosides in a synthetic, modified RNA. Furthermore, in some embodiments of the aspects described herein, a synthetic, modified RNA comprises at least two different modified nucleosides. In some such preferred embodiments of the aspects described herein, the at least two different modified nucleosides are 5-methylcytidine and pseudouridine. A synthetic, modified RNA can also contain a mixture of both modified and unmodified nucleosides.

As used herein, “unmodified” or “natural” nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). In some embodiments, a synthetic, modified RNA comprises at least one nucleoside (“base”) modification or substitution. Modified nucleosides include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine, 2-(aminoalkyl)adenine, 2 (aminopropyl)adenine, 2 (methylthio) N6 (isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6 (methyl)adenine, N6, N6 (dimethyl)adenine, 2-(alkyl)guanine, 2 (propyl)guanine, 6-(alkyl)guanine, 6 (methyl)guanine, 7 (alkyl)guanine, 7 (methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine, 8-(alkenyl)guanine, 8 (alkynyl)guanine, 8-(amino)guanine, 8 (halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine, 8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5 (aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5 (methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine, 5 (trifluoromethyl)cytosine, 6-(azo)cytosine, N4 (acetyl)cytosine, 3 (3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2 (thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil, 4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5 (aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5 (1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5 (methoxycarbonylmethyl)-2-(thio)uracil, 5 (methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil, 5 (trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3 (methyl)uracil, 5-uracil (i.e., pseudouracil), 2 (thio)pseudouracil, 4 (thio)pseudouracil, 2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil, 1 substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1 substituted 2,4-(dithio)pseudouracil, 1 (aminocarbonylethylenyl)-pseudouracil, 1 (aminocarbonylethylenyl)-2(thio)-pseudouracil, 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole, 6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5 substituted pyrimidines, N2-substituted purines, N6-substituted purines, O6-substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Modified nucleosides also include natural bases that comprise conjugated moieties, e.g. a ligand. As discussed herein above, the RNA containing the modified nucleosides must be translatable in a host cell (i.e., does not prevent translation of the polypeptide encoded by the modified RNA). For example, transcripts containing s2U and m6A are translated poorly in rabbit reticulocyte lysates, while pseudouridine, m5U, and m5C are compatible with efficient translation. In addition, it is known in the art that 2′-fluoro-modified bases useful for increasing nuclease resistance of a transcript, leads to very inefficient translation. Translation can be assayed by one of ordinary skill in the art using e.g., a rabbit reticulocyte lysate translation assay.

Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Int. Appl. No. PCT/US09/038,425, filed Mar. 26, 2009; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference in its entirety, and U.S. Pat. No. 5,750,692, also herein incorporated by reference in its entirety.

Another modification for use with the synthetic, modified RNAs described herein involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the RNA. Ligands can be particularly useful where, for example, a synthetic, modified RNA is administered in vivo. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556, herein incorporated by reference in its entirety), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060, herein incorporated by reference in its entirety), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770, each of which is herein incorporated by reference in its entirety), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538, herein incorporated by reference in its entirety), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54, each of which is herein incorporated by reference in its entirety), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783, each of which is herein incorporated by reference in its entirety), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973, herein incorporated by reference in its entirety), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654, herein incorporated by reference in its entirety), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237, herein incorporated by reference in its entirety), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937, herein incorporated by reference in its entirety).

The synthetic, modified RNAs described herein can further comprise a 5′ cap. In some embodiments of the aspects described herein, the synthetic, modified RNAs comprise a 5′ cap comprising a modified guanine nucleotide that is linked to the 5′ end of an RNA molecule using a 5′-5′triphosphate linkage. As used herein, the term “5′ cap” is also intended to encompass other 5′ cap analogs including, e.g., 5′ diguanosine cap, tetraphosphate cap analogs having a methylene-bis(phosphonate) moiety (see e.g., Rydzik, A M et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA 14(6):1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g., Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien, E. et al., (2004) RNA 10(9):1479-1487), or anti-reverse cap analogs (see e.g., Jemielity, J. et al., (2003) RNA 9(9): 1108-1122 and Stepinski, J. et al., (2001) RNA 7(10):1486-1495). In one such embodiment, the 5′ cap analog is a 5′ diguanosine cap. In some embodiments, the synthetic, modified RNA does not comprise a 5′ triphosphate.

The 5′ cap is important for recognition and attachment of an mRNA to a ribosome to initiate translation. The 5′ cap also protects the synthetic, modified RNA from 5′ exonuclease mediated degradation. It is not an absolute requirement that a synthetic, modified RNA comprise a 5′ cap, and thus in other embodiments the synthetic, modified RNAs lack a 5′ cap. However, due to the longer half-life of synthetic, modified RNAs comprising a 5′ cap and the increased efficiency of translation, synthetic, modified RNAs comprising a 5′ cap are preferred herein.

The synthetic, modified RNAs described herein can further comprise a 5′ and/or 3′ untranslated region (UTR). Untranslated regions are regions of the RNA before the start codon (5′) and after the stop codon (3′), and are therefore not translated by the translation machinery. Modification of an RNA molecule with one or more untranslated regions can improve the stability of an mRNA, since the untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation. In addition, modification of an RNA with a 5′ and/or 3′ untranslated region can enhance translational efficiency by binding proteins that alter ribosome binding to an mRNA. Modification of an RNA with a 3′ UTR can be used to maintain a cytoplasmic localization of the RNA, permitting translation to occur in the cytoplasm of the cell. In one embodiment, the synthetic, modified RNAs described herein do not comprise a 5′ or 3′ UTR. In another embodiment, the synthetic, modified RNAs comprise either a 5′ or 3′ UTR. In another embodiment, the synthetic, modified RNAs described herein comprise both a 5′ and a 3′ UTR. In one embodiment, the 5′ and/or 3′ UTR is selected from an mRNA known to have high stability in the cell (e.g., a murine alpha-globin 3′ UTR). In some embodiments, the 5′ UTR, the 3′ UTR, or both comprise one or more modified nucleosides.

In some embodiments, the synthetic, modified RNAs described herein further comprise a Kozak sequence. The “Kozak sequence” refers to a sequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG (SEQ ID NO: 1481), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. The Kozak consensus sequence is recognized by the ribosome to initiate translation of a polypeptide. Typically, initiation occurs at the first AUG codon encountered by the translation machinery that is proximal to the 5′ end of the transcript. However, in some cases, this AUG codon can be bypassed in a process called leaky scanning. The presence of a Kozak sequence near the AUG codon will strengthen that codon as the initiating site of translation, such that translation of the correct polypeptide occurs. Furthermore, addition of a Kozak sequence to a synthetic, modified RNA will promote more efficient translation, even if there is no ambiguity regarding the start codon. Thus, in some embodiments, the synthetic, modified RNAs described herein further comprise a Kozak consensus sequence at the desired site for initiation of translation to produce the correct length polypeptide. In some such embodiments, the Kozak sequence comprises one or more modified nucleosides.

In some embodiments, the synthetic, modified RNAs described herein further comprise a “poly (A) tail”, which refers to a 3′ homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides). The inclusion of a 3′ poly(A) tail can protect the synthetic, modified RNA from degradation in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency. In some embodiments, the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more. In one embodiment, the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides.

It is contemplated that one or more modifications to the synthetic, modified RNAs described herein permit greater stability of the synthetic, modified RNA in a cell. To the extent that such modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the synthetic, modified RNA with the modification, such modifications are specifically contemplated for use herein. Generally, the greater the stability of a synthetic, modified RNA, the more protein can be produced from that synthetic, modified RNA. Typically, the presence of AU-rich regions in mammalian mRNAs tend to destabilize transcripts, as cellular proteins are recruited to AU-rich regions to stimulate removal of the poly(A) tail of the transcript. Loss of a poly(A) tail of a synthetic, modified RNA can result in increased RNA degradation. Thus, in one embodiment, a synthetic, modified RNA as described herein does not comprise an AU-rich region. In particular, it is preferred that the 3′ UTR substantially lacks AUUUA sequence elements.

In one embodiment, a ligand alters the cellular uptake, intracellular targeting or half-life of a synthetic, modified RNA into which it is incorporated. In some embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, intracellular compartment, e.g., mitochondria, cytoplasm, peroxisome, lysosome, as, e.g., compared to a composition absent such a ligand. Preferred ligands do not interfere with expression of a polypeptide from the synthetic, modified RNA.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell targeting agent, (e.g., a lectin, glycoprotein, lipid or protein), or an antibody, that binds to a specified cell type such as a fibroblast cell. A targeting group can be, for example, a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic, among others.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), and transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid).

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a fibroblast cell, or other cell useful in the production of polypeptides. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the synthetic, modified RNA or a composition thereof into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

One exemplary ligand is a lipid or lipid-based molecule. A lipid or lipid-based ligand can (a) increase resistance to degradation, and/or (b) increase targeting or transport into a target cell or cell membrane. A lipid based ligand can be used to modulate, e.g., binding of the modified RNA composition to a target cell.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a host cell. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up, for example, by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Synthesis of Synthetic, Modified RNAs

The synthetic, modified RNAs described herein can be synthesized and/or modified by methods well established in the art, such as those described in “Current Protocols in Nucleic Acid Chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference in its entirety. Transcription methods are described further herein in the Examples.

In one embodiment of the aspects described herein, a template for a synthetic, modified RNA is synthesized using “splint-mediated ligation,” which allows for the rapid synthesis of DNA constructs by controlled concatenation of long oligos and/or dsDNA PCR products and without the need to introduce restriction sites at the joining regions. It can be used to add generic untranslated regions (UTRs) to the coding sequences of genes during T7 template generation. Splint mediated ligation can also be used to add nuclear localization sequences to an open reading frame, and to make dominant-negative constructs with point mutations starting from a wild-type open reading frame. Briefly, single-stranded and/or denatured dsDNA components are annealed to splint oligos which bring the desired ends into conjunction, the ends are ligated by a thermostable DNA ligase and the desired constructs amplified by PCR. A synthetic, modified RNA is then synthesized from the template using an RNA polymerase in vitro. After synthesis of a synthetic, modified RNA is complete, the DNA template is removed from the transcription reaction prior to use with the methods described herein.

In some embodiments of these aspects, the synthetic, modified RNAs are further treated with an alkaline phosphatase.

Plurality of Synthetic, Modified RNAs

In some embodiments of the aspects described herein, a plurality of different synthetic, modified RNAs are contacted with, or introduced to, a cell, population of cells, or cell culture and permit expression of at least two polypeptide products in the cell. In some embodiments, synthetic, modified RNA compositions comprise two or more synthetic, modified RNAs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more synthetic, modified RNAs. In some embodiments, the two or more synthetic, modified RNAs are capable of increasing expression of a desired polypeptide product (e.g., a transcription factor, a cell surface marker, a death receptor, etc.).

In some embodiments, when a plurality of different synthetic, modified RNAs, synthetic, modified RNA compositions, or media comprising a plurality of different synthetic, modified RNAs are used to modulate expression of a desired set of polypeptides, the plurality of synthetic, modified RNAs can be contacted with, or introduced to, a cell, population of cells, or cell culture simultaneously. In other embodiments, the plurality of synthetic, modified RNAs can be contacted with, or introduced to, a cell, population of cells, or cell culture separately. In addition, each synthetic, modified RNA can be administered according to its own dosage regime. For example, in one embodiment, a composition can be prepared comprising a plurality of synthetic, modified RNAs, in differing relative amounts or in equal amounts, that is contacted with a cell such that the plurality of synthetic, modified RNAs are administered simultaneously. Alternatively, one synthetic, modified RNA at a time can be administered to a cell culture (e.g., sequentially). In this manner, the expression desired for each target polypeptide can be easily tailored by altering the frequency of administration and/or the amount of a particular synthetic, modified RNA administered. Contacting a cell with each synthetic, modified RNA separately can also prevent interactions between the synthetic, modified RNAs that can reduce efficiency of expression. For ease of use and to prevent potential contamination, it is preferred to administer to or contact a cell, population of cells, or cell culture with a cocktail of different synthetic, modified RNAs, thereby reducing the number of doses required and minimizing the chance of introducing a contaminant to the cell, population of cells, or cell culture.

The methods and compositions described herein permit the expression of one or more polypeptides to be tuned to a desired level by varying the amount of each synthetic, modified RNA transfected. One of skill in the art can easily monitor the expression level of the polypeptide encoded by a synthetic, modified RNA using e.g., Western blotting techniques or immunocytochemistry techniques. A synthetic, modified RNA can be administered at a frequency and dose that permit a desired level of expression of the polypeptide. Each different synthetic, modified RNA can be administered at its own dose and frequency to permit appropriate expression. In addition, since the synthetic, modified RNAs administered to the cell are transient in nature (i.e., are degraded over time) one of skill in the art can easily remove or stop expression of a synthetic, modified RNA by halting further transfections and permitting the cell to degrade the synthetic, modified RNA over time. The synthetic, modified RNAs will degrade in a manner similar to cellular mRNAs.

Introducing a Synthetic, Modified RNA into a Cell

A synthetic, modified RNA can be introduced into a cell in any manner that achieves intracellular delivery of the synthetic, modified RNA, such that expression of the polypeptide encoded by the synthetic, modified RNA can occur. As used herein, the term “transfecting a cell” refers to the process of introducing nucleic acids into cells using means for facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. As the term is used herein, “transfection” does not encompass viral- or viral particle based delivery methods. Absorption or uptake of a synthetic, modified RNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Further approaches are described herein below or known in the art.

A synthetic, modified RNA can be introduced into a target cell, for example, by transfection, nucleofection, lipofection, electroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)), microinjection (e.g., by direct injection of a synthetic, modified RNA), biolistics, cell fusion, and the like. In an alternative embodiment, a synthetic, modified RNA can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a synthetic, modified RNA (negatively charged polynucleotides) and also enhances interactions at the negatively charged cell membrane to permit efficient cellular uptake. Cationic lipids, dendrimers, or polymers can either be bound to modified RNAs, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases the modified RNA. Methods for making and using cationic-modified RNA complexes are well within the abilities of those skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).

In some embodiments of the aspects described herein, the composition further comprises a reagent that facilitates uptake of a synthetic, modified RNA into a cell (transfection reagent), such as an emulsion, a liposome, a cationic lipid, a non-cationic lipid, an anionic lipid, a charged lipid, a penetration enhancer or alternatively, a modification to the synthetic, modified RNA to attach e.g., a ligand, peptide, lipophillic group, or targeting moiety.

The process for delivery of a synthetic, modified RNA to a cell will necessarily depend upon the specific approach for transfection chosen. One preferred approach is to add the RNA, complexed with a cationic transfection reagent (see below) directly to the cell culture media for the cells.

It is also contemplated herein that a first and second synthetic, modified RNA are administered in a separate and temporally distinct manner. Thus, each of a plurality of synthetic, modified RNAs can be administered at a separate time or at a different frequency interval to achieve the desired expression of a polypeptide. Typically, 100 fg to 100 pg of a synthetic, modified RNA is administered per cell using cationic lipid-mediated transfection. Since cationic lipid-mediated transfection is highly inefficient at delivering synthetic, modified RNAs to the cytosol, other techniques can require less RNA. The entire transcriptome of a mammalian cell constitutes about 1 pg of mRNA, and a polypeptide (e.g., a transcription factor) can have a physiological effect at an abundance of less than 1 fg per cell.

Transfection Reagents

In certain embodiments of the aspects described herein, a synthetic, modified RNA can be introduced into target cells by transfection or lipofection. Suitable agents for transfection or lipofection include, for example, calcium phosphate, DEAE dextran, lipofectin, lipofectamine, DIMRIE C™, Superfect™, and Effectin™ (Qiagen™), Unifectin™, Maxifectin™, DOTMA, DOGS™ (Transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene, poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al., Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88 (1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J. Pharm. 183:195-207 (1999)).

A synthetic, modified RNA can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine™) or non-cationic lipid-based carriers (e.g., Transit-TKOTM™, Mirus Bio LLC, Madison, Wis.). Successful introduction of a modified RNA into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Successful transfection of a modified RNA can also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry.

In some embodiments of the aspects described herein, the synthetic, modified RNA is introduced into a cell using a transfection reagent. Some exemplary transfection reagents include, for example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731). Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a) D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

In other embodiments, highly branched organic compounds, termed “dendrimers,” can be used to bind the exogenous nucleic acid, such as the synthetic, modified RNAs described herein, and introduce it into the cell.

In other embodiments of the aspects described herein, non-chemical methods of transfection are contemplated. Such methods include, but are not limited to, electroporation (methods whereby an instrument is used to create micro-sized holes transiently in the plasma membrane of cells under an electric discharge), sono-poration (transfection via the application of sonic forces to cells), and optical transfection (methods whereby a tiny (˜1 μm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser). In other embodiments, particle-based methods of transfections are contemplated, such as the use of a gene gun, whereby the nucleic acid is coupled to a nanoparticle of an inert solid (commonly gold) which is then “shot” directly into the target cell's nucleus; “magnetofection,” which refers to a transfection method, that uses magnetic force to deliver exogenous nucleic acids coupled to magnetic nanoparticles into target cells; “impalefection,” which is carried out by impaling cells by elongated nanostructures, such as carbon nanofibers or silicon nanowires which have been coupled to exogenous nucleic acids.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols, such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes, such as limonene and menthone.

Synthetic, Modified RNA Compositions

In some embodiments of the aspects described herein, particularly embodiments involving in vivo administration of synthetic, modified RNAs or compositions thereof, the synthetic, modified RNAs described herein are formulated in conjunction with one or more penetration enhancers, surfactants and/or chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.

The compositions described herein can be formulated into any of many possible administration forms, including a sustained release form. In some preferred embodiments of the aspects described herein, formulations comprising a plurality of different synthetic, modified RNAs are prepared by first mixing all members of a plurality of different synthetic, modified RNAs, and then complexing the mixture comprising the plurality of different synthetic, modified RNAs with a desired ligand or targeting moiety, such as a lipid. The compositions can be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

The compositions described herein can be prepared and formulated as emulsions for the delivery of synthetic, modified RNAs. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain further components in addition to the dispersed phases, and the active drug (i.e., synthetic, modified RNA) which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

As noted above, liposomes can optionally be prepared to contain surface groups to facilitate delivery of liposomes and their contents to specific cell populations. For example, a liposome can comprise a surface groups such as antibodies or antibody fragments, small effector molecules for interacting with cell-surface receptors, antigens, and other like compounds.

Surface groups can be incorporated into the liposome by including in the liposomal lipids a lipid derivatized with the targeting molecule, or a lipid having a polar-head chemical group that can be derivatized with the targeting molecule in preformed liposomes. Alternatively, a targeting moiety can be inserted into preformed liposomes by incubating the preformed liposomes with a ligand-polymer-lipid conjugate.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 (Thierry et al.) discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 (Tagawa et al.) discloses protein-bonded liposomes and asserts that the contents of such liposomes can include an RNA molecule. U.S. Pat. No. 5,665,710 (Rahman et al.) describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 (Love et al.) discloses liposomes comprising RNAi molecules targeted to the raf gene. In addition, methods for preparing a liposome composition comprising a nucleic acid can be found in e.g., U.S. Pat. Nos. 6,011,020; 6,074,667; 6,110,490; 6,147,204; 6, 271, 206; 6,312,956; 6,465,188; 6,506,564; 6,750,016; and 7,112,337. Each of these approaches can provide delivery of a synthetic, modified RNA as described herein to a cell.

In some embodiments of the aspects described herein, the synthetic, modified RNA described herein can be encapsulated in a nanoparticle. Methods for nanoparticle packaging are well known in the art, and are described, for example, in Bose S, et al (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J. Virol. 78:8146. 2004); Dong Y et al. Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R. et al (Improved body distribution of 14C-labelled AZT bound to nanoparticles in rats determined by radioluminography. J Drug Target 5:171.1998); Sakuma S R et al (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int J Pharm 177:161. 1999); Virovic L et al. Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2:707.2005); and Zimmermann E et al, Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001), the contents of which are herein incorporated in their entireties by reference.

Methods for Further Avoiding a Cell's Innate Immune or Interferon Response

Importantly, the inventors have discovered that the synthetic, modified RNAs described herein are significantly less cytotoxic when transfected into cells than their synthetic, unmodified RNA counterparts having the same nucleic acid sequence (as measured using e.g., TUNEL assay or simply monitoring cellularity after transfection), which permits repeated transfections of the cells for the duration necessary to express a polypeptide in a cell, or alter the phenotype or developmental fate of the cell. The decrease in cytotoxicity stems, in part, from the presence of modified nucleoside(s) in the RNA, which reduce or prevent the development of a cellular interferon response. In some embodiments of the aspects described herein, the cellular innate immune or interferon response comprises expression of a Type I or Type II interferon. In some embodiments of the aspects described herein, the cellular innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. As noted herein, such modifications for reducing or preventing the cellular innate response include, but are not limited to, 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′ deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In some preferred embodiments, the modifications comprise 5-methylcytidine and pseudouridine.

However, the cells transfected with the synthetic, modified RNA compositions described herein can further be treated or used with other measures to prevent or reduce any remaining cytotoxicity caused by the transfection procedure, the synthetic, modified RNAs, or a combination thereof. The cytotoxicity of synthetic, unmodified RNAs involves a cellular innate immune response designed to recognize a foreign pathogen (e.g., virus) and to produce interferons, which in turn stimulates the activity of the protein kinase PKR, Toll-like receptors (TLRs) and RIG-1, among others, to mediate anti-viral actions. A significant part of an individual cell's innate immune response to foreign RNA is represented by the so-called “PKR response” triggered largely by double-stranded RNA. To the extent that all or part of the PKR response pathway can be activated by foreign single-stranded RNA, such as synthetic, modified RNAs described herein, the response is discussed herein below.

Double stranded RNA dependent protein kinase (PKR) is a member of a family of kinases that phosphorylates the alpha subunit of protein synthesis initiation factor, eIF-2 (eIF-2α) and plays a role in the translational down regulation of gene expression (Clemens et al. Mol. Biol. Rep. 1994; vol. 19: 210-10). Activation of PKR involves two molecules binding in tandem to double stranded RNA and then phosphorylating each other in an intramolecular event. (Wu et al. 1997, J. Biol. Chem 272:1291-1296). PKR has been implicated in processes that rely on apoptosis as control mechanisms in vivo including antiviral activities, cell growth regulation and tumorigenesis (Donze et al. EMBO J. 1995, vol. 14: 3828-34; Lee et al. Virology 1994, vol. 199: 491-6; Jagus et al. Int. J. Biochem. Cell. Biol. 1989, vol. 9: 1576-86). Regulation of protein synthesis through activated PKR arises from the interaction of PKR with foreign RNA.

It has been shown that the PKR response can be reduced by removing the 5′-triphosphate on an RNA molecule, and that RNAs having a 5′-monophosphate, -diphosphate or -7-methyl guanosine cap do not activate PKR. Thus, in one embodiment, the synthetic, modified RNA described herein comprises a 5′-monophosphate, a 5′-diphosphate, or a 5′ 7-methyl guanosine cap to escape the immune response initiated by PKR. In another embodiment, the synthetic, modified RNA as described herein is treated to remove the 5′-triphosphate using an alkaline phosphatase, e.g., calf intestinal phosphatase. Other modifications to prevent activation of the immune response mediators (e.g., PKR, TLRs, and RIG-1) are discussed in detail in Nallagatla, S R, et al., (2008) RNA Biol 5(3):140-144, which is herein incorporated by reference in its entirety.

TLR7 is known to recognize single stranded RNA and binds exogenous RNAs, such as viral single-stranded RNAs in endosomes. Modifications to the RNA that reduce recognition and/or signaling by TLR7 can reduce this aspect of the innate immune response to the RNA. TLR7 signals through MyD88 and can activate a type I IFN pathway as well as an NF-κB/IL-8 pathway.

In one embodiment, the innate immune response or interferon response can be further decreased in cells transfected with a synthetic, modified RNA as described herein by co-transfection of a dominant negative mutant of a protein involved in the immunity pathways, such as RIG-1, MYD88, VISA, PKR and Toll-like receptors. Alternatively, RNA interference (e.g., siRNA, shRNA, etc.) can be used to inhibit expression of RIG-1, MYD88, VISA, PKR, TRIF, TRL7, or TLR8, which will result in a lower innate immune mediated response in the cells.

Another approach to reduce the innate immune mediated response is to inhibit the effect of secreted interferon on cellular receptors, for example, by scavenging secreted interferon using a soluble interferon receptor (e.g., B18R) or a neutralizing antibody. In one embodiment, a modified RNA encoding an interferon scavenging agent (e.g., a soluble interferon receptor) can be administered to cells to further reduce the innate immune response of the cells.

In one embodiment, the cells transfected with synthetic, modified RNA as described herein can be grown with genetically-engineered feeder cells that secrete B18R or neutralizing antibodies to type-1 interferons.

Small molecules that inhibit the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and 2-aminopurine (a PKR inhibitor), can also be administered into the culture media of cells transfected with the synthetic, modified RNAs described herein. Some non-limiting examples of commercially available TLR-signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available for purchase from INVIVOGEN™). In addition, inhibitors of pattern recognition receptors (PRR) (which are involved in innate immunity signaling) such as 2-aminopurine, BX795, chloroquine, and H-89, can also be used in the compositions and methods described herein. Media supplementation with cell-penetrating peptides that inhibit proteins in the immunity pathways described above can also be combined with the use of synthetic, modified RNAs provided herein. Some non-limiting examples of commercially available cell-penetrating peptides include Pepin-MYD (INVIVOGEN™) or Pepinh-TRIF (INVIVOGEN™). An oligodeoxynucleotide antagonist for the Toll-like receptor signaling pathway can also be added to the cell culture media to reduce immunity signaling.

Another method for reducing the immune response of a cell transfected with the synthetic, modified RNAs described herein is to co-transfect mRNAs that encode negative regulators of innate immunity such as NLRX1. Alternatively, one can co-transfect viral proteins known to modulate host cell defenses such as NS1, NS3/4A, or A46R.

In another embodiment, a synthetic, modified RNA composition encoding inhibitors of the innate immune system can be used to avoid the innate immune response generated in the cell.

It is also contemplated herein that, in some embodiments, in a research setting one of skill in the art can avoid the innate immune response generated in the cell by using cells genetically deficient in antiviral pathways (e.g., VISA knockout cells).

Since induction of the innate immune response results in cytokine release and death of the cells in culture, one can determine the extent of activation of an innate immune or interferon response by measuring e.g., apoptosis (using e.g., a TUNEL assay), reduced growth rate, reduced cellularity, reduction in global protein production, or secretion of cytokines (e.g., type-I interferons such as IFN-alpha and IFN-beta, type II interferons, such as IFNγ), or upregulation of interferon stimulated genes or interferon signature genes (e.g., IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. The level of cytokine release or cell death in a transfected cell culture treated with one of the above measures described for further reducing the innate immune response can be compared to the level of an equivalent cell culture not treated to further reduce the innate immune response.

Cell Types

Provided herein are cells contacted with a synthetic, modified RNA molecule encoding a polypeptide, or a progeny cell of the contacted cell, where the synthetic, modified RNA molecule comprises one or more modifications, such that introducing the synthetic, modified RNA molecule to the cell results in a reduced innate immune response relative to the cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising the one or more modifications. In some embodiments of these aspects, at least two nucleosides are modified. In some embodiments of the aspects described herein, the cellular innate immune or interferon response comprises expression of a Type I or Type II interferon. In some embodiments of the aspects described herein, the cellular innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. As described herein, such modifications for reducing or preventing the cellular innate immune response include, but are not limited to, 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′ deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In some preferred embodiments, the modifications comprise 5-methylcytidine and pseudouridine.

Essentially any cell type can be transfected with synthetic, modified RNAs as described herein to alter the phenotype of the cell. Thus, differentiated somatic cells and stem cells, as well of cells of a cell line, can be transfected with synthetic, modified RNA as described herein. Provided herein are exemplary somatic cells, stem cells, and cell line sources useful with the methods and compositions described herein. However, the description herein is not meant to be limiting and any cell known or used in the art can be phenotypically modified by introducing one or more synthetic, modified RNAs as described herein. In embodiments relating to tissue regeneration or transplantation in a subject, the cells can be from an autologous, i.e., from the same subject, or from heterologous sources.

Somatic Cells

Essentially any primary somatic cell type can be used in the preparation of cells with an altered phenotype or altered developmental potential described herein. Some non-limiting examples of primary cells include, but are not limited to, fibroblast, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, immune cells, hepatic, splenic, lung, circulating blood cells, gastrointestinal, renal, bone marrow, and pancreatic cells. The cell can be a primary cell isolated from any somatic tissue including, but not limited to, brain, liver, lung, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc. The term “somatic cell” further encompasses primary cells grown in culture, provided that the somatic cells are not immortalized.

Where the cell is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used, and are known to those of skill in the art. Isolation and culture methods for various cells are well within the abilities of one skilled in the art.

Further, the parental cell can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. In some embodiments, the cell is a human cell. In an alternate embodiment, the cell is from a non-human organism such as a non-human mammal.

Stem Cells

One of the most intriguing aspects of the technologies comprising the synthetic, modified RNAs described herein is the ability to use such synthetic, modified RNAs to both generate a stem cell from a differentiated cell, and to then direct the differentiation of the stem cell to one or more desired cell types.

Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells, depending on their level of differentiation, are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts. (See, e.g., Potten et al., Development 110: 1001 (1990); U.S. Pat. Nos. 5,750,376, 5,851,832, 5,753,506, 5,589,376, 5,824,489, 5,654,183, 5,693,482, 5,672,499, and 5,849,553, all herein incorporated in their entireties by reference). The stem cells for use with the compositions and methods comprising synthetic, modified RNAs described herein can be naturally occurring stem cells or “induced” stem cells generated using the compositions, kits, and methods described herein, or by any method or composition known to one of skill in the art.

It is specifically noted that stem cells are useful not only for exploiting their differentiation potential to make desired cells, but also as a source for high quality iPS cells. That is, a non-pluripotent stem cell can be the starting point for the generation of high quality iPS cells by transfecting the non-pluripotent stem cell with one or more synthetic, modified RNAs encoding reprogramming factors, as described herein.

Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and the cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).

Transfection with synthetic, modified RNAs directing the reprogramming of somatic, differentiated cells to pluripotency is specifically demonstrated herein. However, as also demonstrated herein, transfection with synthetic, modified RNAs can also be used to drive the differentiation, i.e., decrease the developmental potential of stem cells other than iPS cells,

Stem cells of interest for producing cells with a desired phenotype or a reduced differentiation potential include embryonic cells of various types, exemplified by human embryonic stem (hES) cells, described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage committed stem cells, such as hematopoietic or pancreatic stem cells. In some embodiments, the host cell transfected with synthetic, modified RNA is a multipotent stem cell or progenitor cell. Examples of multipotent cells useful in methods provided herein include, but are not limited to, murine embryonic stem (ES-D3) cells, human umbilical vein endothelial (HuVEC) cells, human umbilical artery smooth muscle (HuASMC) cells, human differentiated stem (HKB-II) cells, and human mesenchymal stem (hMSC) cells. An additional stem cell type of interest for use with the compositions and methods described herein are cancer stem cells.

Adult stem cells are generally limited to differentiating into different cell types of their tissue of origin. However, if the starting stem cells are derived from the inner cell mass of the embryo, they can generate many cell types of the body derived from all three embryonic cell types: endoderm, mesoderm and ectoderm. Stem cells with this property are said to be pluripotent. Embryonic stem cells are one kind of pluripotent stem cell. Thus, pluripotent embryonic stem cells can be differentiated into many specific cell types, and that differentiation can be driven by the expression of polypeptides from synthetic, modified RNAs as described herein. Since the embryo is a potential source of all types of precursor cells, it is possible to differentiate embryonic stem cells into other lineages by providing the appropriate signals, such as the expression of proteins from synthetic, modified RNAs, to embryonic stem cells. Somatic stem cells also have major advantages, for example, using somatic stem cells allows a patient's own cells to be expanded in culture and then re-introduced into the patient. In addition and importantly, iPS cells generated from a patient provide a source of cells that can be expanded and re-introduced to the patient, before or after stimulation to differentiate to a desired lineage or phenotype. It is also contemplated that the compositions, methods and kits comprising the synthetic, modified RNAs described can be used to alter the developmental potential of a cancer stem cell, and thus render that cancer cell non-cancerous.

Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from a stem cell bank or other recognized depository institution. Other means of producing stem cell lines include the method of Chung et al (2006) which comprises taking a blastomere cell from an early stage embryo prior to formation of the blastocyst (at around the 8-cell stage). The technique corresponds to the pre-implantation genetic diagnosis technique routinely practiced in assisted reproduction clinics. The single blastomere cell is then co-cultured with established ES-cell lines and then separated from them to form fully competent ES cell lines.

Cells can also be derived from human umbilical cord blood cells (HUCBC), which are recognized as a rich source of hematopoietic and mesenchymal stem cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Cord blood cells are used as a source of transplantable stem and progenitor cells and as a source of marrow repopulating cells for the treatment of malignant diseases (e.g. acute lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and nueroblastoma) and non-malignant diseases such as Fanconi's anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). One advantage of HUCBC for use with the methods and compositions described herein is the immature immunity of these cells, which is very similar to fetal cells, and thus significantly reduces the risk for rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497).

In other embodiments of the aspects described herein, cancer stem cells are used with the synthetic, modified RNAs described herein, in order to, for example, differentiate or alter the phenotype of a cancer stem cell to a non-tumorigenic state. It has been recently discovered that stem-like cells are present in some human tumors and, while representing a small minority of the total cellular mass of the tumor, are the subpopulation of tumor cells responsible for growth of the tumor. In contrast to normal stem cells, “tumor stem cells” or “cancer stem cells” are defined as cells that can undergo self-renewal, as well as abnormal proliferation and differentiation to form a tumor. Functional features of tumor stem cells are that they are tumorigenic; they can give rise to additional tumorigenic cells by self-renewal; and they can give rise to non-tumorigenic tumor cells. As used herein, particularly in reference to an isolated cell or isolated cell population, the term “tumorigenic” refers to a cell derived from a tumor that is capable of forming a tumor, when dissociated and transplanted into a suitable animal model such as an immunocompromised mouse. The developmental origin of tumor stem cells can vary among different types of cancers. It is believed, without wishing to be bound or limited by theory, that tumor stem cells may arise either as a result of genetic damage that deregulates normal mechanisms of proliferation and differentiation of stem cells (Lapidot et al., Nature 367(6464): 645-8 (1994)), or by the dysregulated proliferation of populations of cells that acquire stem-like properties.

Tumors contain a distinct subset of cells that share the properties of normal stem cells, in that they proliferate extensively or indefinitely and that they efficiently give rise to additional solid tumor stem cells. Within an established tumor, most cells may have lost the ability to proliferate extensively and form new tumors, while tumor stem cells proliferate extensively and give rise to additional tumor stem cells as well as to other tumor cells that lack tumorigenic potential. An additional trait of tumor stem cells is their resistance to therapeutics, such as chemotherapy. It is the small fraction of tumor stem cells and their immediate daughter cell population that proliferates and ultimately proves fatal.

Examples of tumors from which samples containing cancer stem cells can be isolated from or enriched, for use with the compositions and methods described herein, include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, mesothelioma, Ewing's tumor, lymphangioendotheliosarcoma, synovioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, astrocytic tumors (e.g., diffuse, infiltrating gliomas, anaplastic astrocytoma, glioblastoma, gliosarcoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglial tumors and mixed gliomas (e.g., oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligoastrocytoma), ependymal tumors (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), choroid plexus tumors, neuroepithelial tumors of uncertain origin (astroblastoma, chordoid glioma, gliomatosis cerebri), neuronal and mixed-neuronal-glial tumors (e.g., ganglioglioma and gangliocytoma, desmoplastic infantile astrocytoma and ganglioglioma, dysembryoplastic neuroepithelial tumor, central neurocytoma, cerebellar liponeurocytoma, paraganglioglioma), pineal parenchymal tumors, embryonal tumors (medulloepithelioma, ependymoblastoma, medulloblastoma, primitive neuroectodemmal tumor, atypical teratoid/rhabdoid tumor), peripheral neuroblastic tumors, tumors of cranial and peripheral nerves (e.g., schwannoma, neurinofibroma, perineurioma, malignant peripheral nerve sheath tumor), meningeal tumors (e.g., meningeomas, mesenchymal, non-meningothelial tumors, haemangiopericytomas, melanocytic lesions), germ cell tumors, tumors of the sellar region (e.g., craniopharyngioma, granular cell tumor of the neurohypophysis), hemangioblastoma, melanoma, and retinoblastoma. Additionally, the stem cell isolation methods of the invention are applicable to isolating stem cells from tissues other than characterized tumors (e.g., from tissues of diseases such as the so called “stem cell pathologies”).

Stem cells may be obtained from any mammalian species, e.g. human, primate, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, etc. Embryonic stem cells are considered to be undifferentiated when they have not committed to a specific differentiation lineage. Such cells display morphological characteristics that distinguish them from differentiated cells of embryo or adult origin. Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli.

In some embodiments, the stem cell is isolated. Most conventional methods to isolate a particular stem cell of interest involve positive and negative selection using markers of interest. For example, agents can be used to recognize stem cell markers, for instance labeled antibodies that recognize and bind to cell-surface markers or antigens on desired stem cells can be used to separate and isolate the desired stem cells using fluorescent activated cell sorting (FACS), panning methods, magnetic particle selection, particle sorter selection and other methods known to persons skilled in the art, including density separation (Xu et al. (2002) Circ. Res. 91:501; U.S. patent application Ser. No. 20030022367) and separation based on other physical properties (Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32:839-851).

In those embodiments involving cancer stem cells, cancer stem cells can be identified using cell surface markers that also identify normal stem cells in the tissue of origin. As a non-limiting example, leukemic stem cells (LSCs) express the CD34 surface marker and lack the CD38 surface antigen, as is the case for normal (i.e., non-leukemic) hematopoietic stem cells (Bonnet and Dick, 1997). Cancer stem cells identified by cell surface marker expression can be purified by methods known to one of skill in the art, such as fluorescence-activated cell sorting (FACS). Methods of isolating cancer stem cells can be found in United States Patent Application 20100003265, the contents of which are herein incorporated in their entirety by reference.

Alternatively, genetic selection methods for isolating stem cells can be used, where a stem cell can be genetically engineered to express a reporter protein operatively linked to a tissue-specific promoter and/or a specific gene promoter, therefore the expression of the reporter can be used for positive selection methods to isolate and enrich the desired stem cell. For example, a fluorescent reporter protein can be expressed in the desired stem cell by genetic engineering methods to operatively link the marker protein to a promoter active in a desired stem cell (Klug et al. (1996) J. Clin. Invest. 98:216-224; U.S. Pat. No. 6,737,054). Other means of positive selection include drug selection, for instance as described by Klug et al., supra, involving enrichment of desired cells by density gradient centrifugation. Negative selection can be performed, selecting and removing cells with undesired markers or characteristics, for example fibroblast markers, epithelial cell markers etc.

Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and whose polypeptide products can be used as markers for negative selection. For example, see U.S. application Ser. No. 2003/0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra., each herein incorporated by reference. Human ES cell lines express cell surface markers that characterize undifferentiated nonhuman primate ES and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-I-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globo-series glycolipid Gb5, which carries the SSEA-3 epitope. Thus, GL7 reacts with antibodies to both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines do not stain for SSEA-1, but differentiated cells stain strongly for SSEA-1. Methods for proliferating hES cells in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.

In some embodiments, the methods further provide for enrichment and isolation of stem cells. The stem cells are selected for a characteristic of interest. In some embodiments, a wide range of markers may be used for selection. One of skill in the art will be able to select markers appropriate for the desired cell type. The characteristics of interest include expression of particular markers of interest, for example specific subpopulations of stem cells and stem cell progenitors will express specific markers.

In some embodiments, the stem cells used with the compositions and methods described herein are expanded. The cells are optionally collected, separated, and further expanded generating larger populations of progenitor cells for use in making cells of a particular cell type or cells having a reduced differentiation potential.

Cell Lines

In some embodiments, the cells used with the synthetic, modified RNAs described herein are cells of a cell line. In one such embodiment, the host cell is a mammalian cell line. In one such embodiment, the mammalian cell line is a human cell line.

Examples of human cell lines useful in methods provided herein include, but are not limited to, 293T (embryonic kidney), BT-549 (breast), DMS 114 (small cell lung), DU145 (prostate), HT-1080 (fibrosarcoma), HEK 293 (embryonic kidney), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), HL-60(TB) (leukemia), HS 578T (breast), HT-29 (colon adenocarcinoma), Jurkat (T lymphocyte), M14 (melanoma), MCF7 (mammary), MDA-MB-453 (mammary epithelial), PERC6® (E1-transformed embryonal retina), RXF 393 (renal), SF-268 (CNS), SF-295 (CNS), THP-1 (monocyte-derived macrophages), TK-10 (renal), U293 (kidney), UACC-257 (melanoma), and XF 498 (CNS).

Examples of rodent cell lines useful in methods provided herein include, but are not limited to, mouse Sertoli (TM4) cells, mouse mammary tumor (MMT) cells, rat hepatoma (HTC) cells, mouse myeloma (NS0) cells, murine hybridoma (Sp2/0) cells, mouse thymoma (EL4) cells, Chinese Hamster Ovary (CHO) cells and CHO cell derivatives, murine embryonic (NIH/3T3, 3T3 L1) cells, rat myocardial (H9c2) cells, mouse myoblast (C2C12) cells, and mouse kidney (miMCD-3) cells.

Examples of non-human primate cell lines useful in methods provided herein include, but are not limited to, monkey kidney (CVI-76) cells, African green monkey kidney (VERO-76) cells, green monkey fibroblast (Cos-1) cells, and monkey kidney (CVI) cells transformed by SV40 (Cos-7). Additional mammalian cell lines are known to those of ordinary skill in the art and are catalogued at the American Type Culture Collection catalog (ATCC®, Mamassas, Va.).

Other Cell Types

While mammalian cells are preferred, in some embodiments, the host cell transfected with a modified RNA is a plant cell, such as a tobacco plant cell.

In some embodiments, the transfected cell is a fungal cell, such as a cell from Pichia pastoris, a Rhizopus cell, or a Aspergillus cell.

In some embodiments, the transfected cell is an insect cell, such as SF9 or SF-21 cells from Spodoptera frugiperda or S2 cells from Drosophila melanogaster.

Cell Culture Methods

In general, cells useful with the methods described herein can be maintained and/or expanded in a culture medium that is available to and well-known in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle's Minimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco's Medium®, RPMI-1640 Medium®, and serum-free medium for culture and expansion of progenitor cells SFEM®. Many media are also available as low-glucose formulations, with or without sodium.

Cells can be cultured in low-serum or serum-free “defined” culture medium. Serum-free medium used to culture cells is described in, for example, U.S. Pat. No. 7,015,037. Many cells have been grown in serum-free or low-serum medium. For example, the medium can be supplemented with one or more growth factors. Commonly used growth factors include, but are not limited to, bone morphogenic protein, basic fibroblast growth factor, platelet-derived growth factor and epidermal growth factor, Stem cell factor, and thrombopoietin. See, for example, U.S. Pat. Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161; 6,617,159; 6,372,210; 6,224,860; 6,037,174; 5,908,782; 5,766,951; 5,397,706; and 4,657,866; all incorporated by reference herein for teaching growing cells in serum-free medium.

Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components. Progenitor cells may require additional factors that encourage their attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and vitronectin. Progenitor cells can also be cultured in low attachment flasks such as but not limited to Corning Low attachment plates.

In some embodiments, the host cells are suitable for growth in suspension cultures. Suspension-competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of attachment-dependent cells (e.g., epithelial cells, fibroblasts).

In some embodiments, the host cell is an attachment dependent cell which is grown and maintained in adherent culture.

Altering Cellular Phenotypes and Developmental Potentials

The compositions and methods comprising the synthetic, modified RNAs described herein permit long-term, safe, and efficient alteration of cellular phenotypes or cellular developmental potentials, without the risk of permanent genomic alterations. Such compositions and methods are useful for a variety of applications, indications, and modalities, including, but not limited to, gene therapy, regenerative medicine, cancer therapies, tissue engineering, and drug screening.

Accordingly, provided herein are cells contacted with a synthetic, modified RNA molecule encoding a polypeptide, or a progeny cell of the contacted cell, where expression of the encoded polypeptide in the contacted cell alters a function or a developmental phenotype or developmental potential of the cell, and results in a reduced innate immune response relative to the cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising any modifications. In some embodiments, the developmental potential of the contacted cell is decreased. In some embodiments, the developmental potential of the contacted cell is increased. As such, the polypeptide encoded by the synthetic, modified RNA molecule can be a reprogramming factor, a differentiation factor, or a de-differentiation factor.

Also provided herein are cells comprising an exogenously introduced modified, synthetic RNA encoding a developmental potential altering factor. In some embodiments, the cell is a human cell. In some embodiments of these aspects, the cells or immediate precursor cell(s) have been subjected to at least 3 separate rounds of contacting with the modified, synthetic RNA encoding the developmental potential altering factor. In some such embodiments, the cells have a reduced expression of a Type I or Type II IFN relative to a cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the developmental potential altering factor. In some such embodiments, the cell has a reduced expression of at least one IFN-signature gene relative to a human cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the developmental potential altering factor. As described herein, the IFN-signature gene can be selected from the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. The polypeptide encoded by the exogenous synthetic, modified RNA molecule can be a reprogramming factor, a differentiation factor, or a de-differentiation factor. The cell or its immediate precursor cell(s) can be derived from a somatic cell, a partially reprogrammed somatic cell, a pluripotent cell, a multipotent cell, a differentiated cell, or an embryonic cell.

As used herein, the term “developmental potential of a cell” refers to the total of all developmental cell fates or cell types that can be achieved by a cell upon differentiation. It should be understood that the developmental potential of a cell represents a spectrum: a terminally differentiated cell, e.g., a cardiac myocyte, has essentially no developmental potential under natural conditions—that is, under normal circumstances, it cannot differentiate to another cell type; while at the other end of the spectrum, a totipotent embryonic stem cell has the potential to differentiate to or give rise to cells of every type in an organism, as well as the extra-embryonic structures. A cell with “parental developmental potential” refers to a cell having the developmental potential of the parent cell that gave rise to it.

The term “developmental potential of a cell” is relative. For example, where a stem cell undergoes differentiation to a more differentiated or specialized phenotype, the resulting cell has a reduced developmental potential relative to the stem cell that produced it. Unless specifically stated otherwise, the developmental potential of a cell is the potential it has assuming no further manipulation of its potential—that is, while it is acknowledged that the technology is available (as described herein) to artificially increase, decrease or otherwise alter the developmental potential of nearly any cell, to say that a cell has “reduced developmental potential” means that, without further artificial manipulation to force the cell to a less differentiated phenotype, the cell can give rise to at least one fewer cell types than its immediate predecessor cell. That is, the cell resulting from a differentiation event has a reduced developmental potential despite the fact that it could possibly be manipulated to again become less differentiated. Thus, a cell with greater or higher developmental potential can differentiate into a greater variety of different cell types than a cell having a lower or decreased developmental potential.

Where, for example, a terminally- or only partially-differentiated cell is induced by artificial manipulation to become an induced pluripotent stem cell (an iPS cell), the resulting cell has increased developmental potential relative to the cell that produced it. As used herein, a “change” or “alteration” in the developmental potential of a cell occurs when the range of phenotypes to which a given cell can differentiate or give rise increases or decreases relative to the range naturally available to the cell prior to a differentiation, dedifferentiation or trans-differentiation event. By “increase” in this context is meant that there is at least additional one cell type or lineage to which a given cell can differentiate relative to the potential of the starting cell. By “decrease” in this context is meant that there is at least one fewer cell type or lineage to which the given cell can differentiate or give rise, relative to the potential of the starting cell.

Methods of manipulating the developmental potential of a cell, both to increase the potential and to decrease it, are described herein and others are known in the art. A “change” or “alteration” in the developmental potential of a cell can occur naturally, where, for example, a cell differentiates to a more specialized phenotype in its native environment in vivo. In various preferred aspects described herein, developmental potential or cell fate are directed by outside manipulation, and preferably by transfection with synthetic, modified RNA, as that term is defined herein. Thus, in one aspect, cells are contacted or transfected with synthetic, modified RNAs encoding one or more factors that re-direct or modify the phenotype of the cells.

Synthetic, modified RNAs as described herein can be made that direct the expression of essentially any gene product whose coding sequences can be cloned. The expression of the gene product from synthetic, modified RNA introduced to a cell that does not normally express that gene product necessarily results in a change in the phenotype of the cell whether or not it changes the differentiation status or differentiation potential of the cell. Simply put, the new phenotype is the cell's expression of the new gene product. Thus, in one aspect, encompassed herein is the expression of a protein from a synthetic, modified RNA introduced to a cell. Expression that does not necessarily change the differentiation status of the cell can nonetheless be useful in such embodiments, for example, where one wishes to correct or replace a defective function in a cell, due to a genetic defect or polymorphism, or in embodiments to target a cell to a particular location, e.g., by expressing a receptor or where one wishes to induce cell death in e.g., a tumor by expressing a death receptor, a death ligand, a cell cycle inhibitor etc.

In other aspects, the synthetic, modified RNAs described herein are well suited for directing the expression of any gene sequence, but are particularly well suited for modifying the differentiation status or the developmental potential of a cell, and for doing so without permanent change to the genome of the cell. This is true in part because reprogramming, differentiation and transdifferentiation each require relatively prolonged expression of one or more polypeptide factors in a target cell. Non-modified RNA is recognized as foreign by the cell's innate immune defenses against viral and bacterial RNA. If the cell transfected with non-modified RNA is not induced to undergo apoptosis or to otherwise shut down protein synthesis by a first transfection event, it will likely do so upon a subsequent transfection event with unmodified RNA.

Reprogramming

The production of cells having an increased developmental potential (e.g., iPS cells) is generally achieved by the introduction of nucleic acid sequences, specifically DNA, encoding stem cell-associated genes into an adult, somatic cell. Historically, these nucleic acids have been introduced using viral vectors and the expression of the gene products results in cells that are morphologically, biochemically, and functionally similar to pluripotent stem cells (e.g., embryonic stem cells). This process of altering a cell phenotype from a somatic cell phenotype to a pluripotent stem cell phenotype is termed “reprogramming.” In the reprogramming methods described herein, the reprogramming is achieved by repeated transfection with synthetic, modified RNAs encoding the necessary reprogramming factors. The repeated transfection provides prolonged expression of the factors encoded by the synthetic, modified RNAs necessary to shift the developmental potential of the cell.

Accordingly, provided herein are pluripotent cells that are not embryonic stem cells, and which were not induced by viral expression of one or more reprogramming factors, and which when subjected to an unsupervised hierarchical cluster analysis, cluster more closely to embryonic stem cells than do pluripotent cells induced by viral expression of one or more reprogramming factors, exogenous protein introduction of one or more reprogramming factors, small molecule mediated expression or induction of one or more reprogramming factors, or any combination thereof. In some aspects, provided herein are pluripotent cells that are not embryonic stem cells, and which were not induced by viral expression of one or more reprogramming factors. In such aspects, the pluripotent cell subjected to an unsupervised hierarchical cluster analysis clusters more closely to a human embryonic stem cell than does a pluripotent cell induced by viral expression of one or more reprogramming factors. The pluripotent cell is generated from a precursor somatic cell, such as a precursor human somatic cell. The pluripotent cell or its immediate precursor cell(s) can also be derived from a somatic cell, partially reprogrammed somatic cell, a pluripotent cell, a multipotent cell, a differentiated cell, or an embryonic cell.

Reprogramming to generate pluripotent cells, as described herein, can be achieved by introducing a one or more synthetic, modified RNAs encoding stem cell-associated genes including, for example Oct-4 (also known as Oct-3/4 or Pouf51) (SEQ ID NO: 788), Sox1, Sox2 (SEQ ID NO: 941 or SEQ ID NO: 1501), Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4 (SEQ ID NO: 501), Klf5, NR5A2, c-Myc (SEQ ID NO: 636), 1-Myc, n-Myc, Rem2, Tert, LIN28 (SEQ ID NO: 524), and Sall4.

Accordingly, in some embodiments, the reprogramming factor is selected from the group consisting of: OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28. In general, successful reprogramming is accomplished by introducing at least Oct-4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell. In some embodiments, LIN28 is also introduced. The generation of iPS cells using transfection of the synthetic, modified RNAs described herein, also referred to herein as “RiPS,” from a variety of starting cell types, including an adult somatic cell, is demonstrated in the Examples herein. The generation of reprogrammed cells using the compositions and methods described herein preferably causes the induction of endogenous stem-cell associated genes, such as SOX2, REX1, DNMT3B, TRA-1-60, TRA-1-81, SSEA3, SSEA4, OCT4, and NANOG. In some embodiments, at least two endogenous stem-cell-associated genes are induced. Preferably, the endogenous expression is at a level comparable to an embryonic stem cell, such as an embryonic stem cell cultured within the same laboratory.

The methods to reprogram cells using the synthetic, modified RNAs described herein can involve repeated contacting of the cells, such as somatic cells, in order to permit sufficient expression of the encoded reprogramming factors to maintain a stable change in the developmental potential of the cells, or progeny cells thereof, being contacted. Such methods can involve repeated transfections, such as for example, at least two, at least five, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more transfections. In other words, the methods comprise repeating transfection using the synthetic, modified RNAs until a desired phenotype of the cell or population of cells is achieved. In some embodiments, the methods further comprise contacting with or introducing the reprogramming factors to the cells under low-oxygen conditions.

The efficiency of reprogramming (i.e., the number of reprogrammed cells) can be enhanced by the addition of various small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135, which are incorporated herein by reference in their entirety. It is contemplated that the methods described herein can also be used in combination with a single small molecule (or a combination of small molecules) that enhances the efficiency of induced pluripotent stem cell production or replaces one or more reprogramming factors during the reprogramming process. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), and trichostatin (TSA), among others.

In some embodiments of the aspects described herein, an inhibitor of p53 can be used to reduce the stress response during a reprogramming regimen to direct the cell fate away from an apoptotic stimulus and towards reprogramming. Thus, treatment with a p53 inhibitor can enhance reprogramming in a population of cells. In one such embodiment, the inhibitor of p53 comprises an siRNA directed against p53 that is administered or expressed in the reprogramming cell. In another embodiment, a small molecule inhibitor of p53 (e.g., pifithrin-α) is administered to cells during the reprogramming process. In one embodiment, a modified RNA encoding Bcl2 is administered to the cells prior to, or in conjunction with, a modified RNA composition encoding at least one reprogramming factor to prevent apoptosis of cells during the process of reprogramming.

To confirm the induction of pluripotent stem cells, isolated clones can be tested for the expression of an endogenous stem cell marker. Such expression identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides. Further evidence of reprogramming is shown by a reduction in or the loss of lamin A/C protein expression. Alternatively, reprogramming is detected by measuring an increase in acetylation, such as increased acetylation of H3 and H4 within the promoter of Oct4, or by measuring a decrease in methylation, for example, by measuring the demethylation of lysine 9 of histone 3. In each of these cases, reprogramming is measured relative to a control cell. In other embodiments, reprogramming is assayed by any other method that detects chromatin remodeling leading to the activation of an embryonic stem cell marker, such as Oct4.

The pluripotent stem cell character of the isolated cells can be confirmed by any of a number of tests evaluating the expression of ES markers and the ability to differentiate to cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells are introduced to nude mice and histology and/or immunohistochemistry is performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells.

The pluripotent cells generated using the compositions and methods comprising the synthetic, modified RNAs described herein cluster more closely to a human embryonic stem cell than do pluripotent cells induced by viral expression of one or more reprogramming factors, when subjected to an unsupervised hierarchical analysis, i.e., the pluripotent cells have a phenotype closer to a embryonic stem cell phenotype than do pluripotent cells induced by viral expression of one or more reprogramming factors. In some embodiments, the unsupervised hierarchical cluster analysis is performed using a Euclidean distance with average linkage method in which the similarity metric for comparison between different cells is indicated on the height of cluster dendrogram. The unsupervised hierarchical cluster analysis can be performed on any data set available to a skilled artisan, such as gene expression data, protein expression data, DNA methylation data, histone modification data, and microRNA data.

Clustering, including, “unsupervised clustering analysis” or “unsupervised cluster analysis” refers to methods used in multivariate analysis to divide up objects into similar groups, or, in some embodiments, groups whose members are all close to one another on various dimensions being measured in the various objects. A key component of the analysis is repeated calculation of distance measures between objects, and between clusters once objects begin to be grouped into clusters. The outcome is typically represented graphically as a dendrogram. Hierarchical cluster analysis can be performed using any of a variety of unbiased computational methods, algorithms and software programs known to one of skill in the art that identify clusters or natural data structures from large data sets, such as, for example, gene expression data sets. Such methods include, but are not limited to, bottom-up hierarchical clustering, K-means clustering Affinity Propagation, non-Negative Matrix Factorization, spectral clustering, Self-Organizing Map (SOM) algorithms, and the like. In some embodiments of the aspects described herein, one SOM-based method for use in unsupervised hierarchical clustering analysis of cells contacted with the synthetic, modified RNAs described herein is the Automatic clustering using density-equalized SOM Ensembles (AUTOsome) method as described in A. M. Newman and J. B. Cooper (2010, Cell Stem Cell, 7:258-262) and A. M. Newman and J. B. Cooper (2010, BMC Bioinformatics 2010, 11:117), the contents of each of which are herein incorporated in their entireties by reference.

Accordingly, also provided herein are compositions for generating such pluripotent cells, comprising at least one synthetic, modified RNA encoding a reprogramming factor, and cell growth media. The synthetic, modified RNAs can comprise any modification for reducing the innate immune response, as described herein, such as a 5′ cap, a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. In preferred embodiments, the synthetic, modified RNAs comprise at least two nucleoside modifications, preferably 5-methylcytidine (5mC) and pseudouridine.

In some embodiments, the compositions permit an efficiency of pluripotent cell generation from a starting population of cells, such as somatic cells, of at least 1%. In some embodiments, the efficiency of pluripotent cell generation is at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2.0%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3.0%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4.0%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5.0%, 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6.0%, 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 7.0%, 7.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9.0%, 9.1%, at least 9.2%, at least 9.3%, at least 9.4%, at least 9.5%, at least 1.6%, at least 9.7%, at least 9.8%, at least 9.9%, at least 10.0%, or more.

In some embodiments, the compositions permit a rate of pluripotent cell generation from a starting population of cells, such as somatic cells of less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, and greater than 7 days.

The reprogramming factor(s) for use in the compositions, methods, and kits for reprogramming cells described herein is selected from the group consisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some embodiments, the compositions comprise at least 4 synthetic, modified RNAs encoding at least 4 different reprogramming factors. In some such embodiments, the at least 4 different reprogramming factors encoded by the at least 4 modified synthetic RNAs comprise OCT4, SOX2, KLF4, and c-MYC. The compositions can further comprise a modified synthetic RNA encoding a LIN28 reprogramming factor. In some embodiments, the composition does not comprise a modified, synthetic RNA encoding the reprogramming factor c-MYC.

Transdifferentiation

Transdifferentiation refers to a process by which the phenotype of a cell can be switched to that of another cell type, without the formation of a pluripotent intermediate cell. Thus, the methods do not require that the cell first be de-differentiated (or reprogrammed) and then differentiated to another cell type; rather the cell type is merely “switched” from one cell type to another without first forming a less differentiated phenotype. Thus, “transdifferentiation” refers to the capacity of differentiated cells of one type to lose identifying characteristics and to change their phenotype to that of other fully differentiated cells.

Transdifferentiation can be achieved by introducing into a cell a synthetic, modified RNA composition that permits expression of a cell-type specific differentiation factor. For example, to transdifferentiate a cell to a myogenic lineage one can express MyoD using a modified RNA as described herein. While the introduction of a single differentiation factor can be enough to transdifferentiate a cell, it is also contemplated herein that a plurality of different differentiation factors are introduced to the cell during the transdifferentiation regime. Alternatively, synthetic, modified RNAs that inhibit expression of cell-type specific polypeptides of the original cell-type can also be introduced to the cell, in effect “turning off” the original phenotype of the cell. In one embodiment, modified RNAs that express a desired cell-type specific polypeptide to turn on a desired phenotype are used in combination with modified RNA interference molecules used to turn off the existing cell phenotype, in order to cause transdifferentiation of the cell from one phenotype to another.

Transdifferentiation can be useful in tissue engineering at e.g., an injury or disease site. In one embodiment, transdifferentiation is performed in vivo at the site of injury or disease. In another embodiment, an organ or tissue can be transdifferentiated/regenerated in vitro, and then introduced back into the body.

Differentiation

Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell (e.g., a terminally differentiated cell) such as, for example, a cardiomyocyte, a nerve cell or a skeletal muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell (e.g., reduced differentiation potential). The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell (i.e., increased developmental potential). As used herein, the lineage of a cell defines the heredity or fate of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.

Cells that are differentiated using the compositions and methods comprising synthetic, modified RNAs, as described herein, can be differentiated into any cell type or lineage known to one of skill in the art. Such cells can be of a lineage selected from an ecotodermal lineage, a mesodermal lineage, or an endodermal lineage. Exemplary ectodermal lineage cells include, but are not limited to, cells of the epidermis (skin cells, melanocytes), and cells of the neuronal lineage. Exemplary mesodermal lineage cells include, but are not limited to, cells of the circulatory system (cardiac cells and blood vessel cells), cells of the connective tissue, bone cells, dermal cells, myocytes (smooth and skeletal), certain cells of the urinary system, such as kidney cells, splenic cells, mesothelial cells (cells of the peritoneum, pleura, and pericardium), non-germ cells of the reproductive system, and hematopoietic lineage cells. Exemplary endodermal lineage cells include, but are not limited to, cells of the gastrointestinal system, cells of the respiratory tract, cells of the endocrine glands, cells of the auditory system, and certain cells of the urinary system, such as the bladder and parts of the urethra.

Accordingly, compositions and methods described herein include a method for programming or directing the differentiation of cells (e.g., stem cells) comprising contacting the cells desired to be differentiated with a synthetic, modified RNA or synthetic, modified RNA composition. The cells can be transfected a plurality of times until the desired differentiated phenotype is achieved, as measured by e.g., a gene expression array of cell-type specific markers, Western blotting, cell function assays etc. A selection compound may be added to the mixture, but is not required.

Typically, the synthetic, modified RNA composition transfected into the cells to promote their differentiation encodes a cell-type specific differentiation factor or factors. For example, to differentiate a cell to a neuronal cell phenotype, a synthetic, modified RNA encoding at least one neuronal differentiation factor, for example Asc11, Brn2, Myt11, or a combination thereof is transfected into the cell. To promote differentiation to a myogenic phenotype, a synthetic, modified RNA such as one encoding MyoD can be transfected into a cell. To differentiate a cell to a macrophage phenotype, a macrophage factor such as e.g., CEBP-alpha or PU.1 is transfected into the cell. In one embodiment, a modified RNA that encodes Ngn3, Pdx1, MAFA, or any combination thereof can be used to differentiate cells to a pancreatic beta cell phenotype. A synthetic, modified RNA encoding PRDM16 can be applied to Myf5-expressing progenitors to induce differentiation into brown fat cells. Oligodendrocytes may be specified from neural precursors using a synthetic, modified RNA encoding Asc11. It has been reported that hepatocyte differentiation requires the transcription factor HNF-4α. (Li et al., Genes Dev. 14:464, 2000). A synthetic, modified RNA can be applied to a cell, such as a stem cell or induced pluripotent stem cell generated using the compositions described herein, that inhibit or suppress one or more component of the wnt/β-catenin pathway to become a cardiovascular progenitor cell. These examples are not meant to be limiting and essentially any cell-type specific factor or differentiation factor known in the art can be expressed in a cell using a synthetic, modified RNA or synthetic, modified RNA composition as described herein. Table 1 provides a non-limiting list of exemplary transcription factors and corresponding mRNA sequence identifiers that can be used to alter the developmental potential or phenotype of a cell.

In other embodiments, cells with higher or increased developmental potential, e.g., pluripotent cells, multipotent cells, etc., can be induced to differentiate by manipulating their external environment. For example, cells can be maintained under culture conditions that induce differentiation of the cells to a desired lineage. As but one example, in some embodiments, cells with higher or increased developmental potential, generated using the compositions and methods comprising synthetic, modified RNAs described herein, can be differentiated into islet-like cells for administration to a patient in need thereof, for example, a patient having or at risk for diabetes. In such embodiments, islet-like cells, which includes insulin-producing cells and glucagon-producing cells, can be differentiated using any of the methods described in US Patent Publication No.: 20100240130, the contents of which are herein incorporated in their entirety by reference. For example, cells can be differentiated whereby the first culturing step takes place in the presence of an Activin, the next culturing step utilizes a suspension culture that takes place in the presence of a noggin, an FGF-2, and an EGF, and a final culturing step in which the cells are cultured with nicotinamide. In certain embodiments, sodium butyrate can be included in the culture medium. In other embodiments, pluripotent cells can be differentiated into islet-like cells by directed differentiation. In certain embodiments, expression of additional genes at the site of islet-like cell administration, using the compositions and methods described herein, can facilitate adoption of the functional β-islet cell phenotype, enhance the beneficial effect of the administered cells, and/or increase proliferation and/or activity of host cells neighboring the treatment site.

In other embodiments, cells with higher or increased developmental potential, generated using the compositions and methods comprising synthetic, modified RNAs described herein, can be differentiated, for example, into neuronal cells, such as oligodendrocytes, for example, for treatment of spinal cord injuries. In such embodiments, pluripotent cells can be differentiated using any of the compositions or methods found in US Patent Publication No.: 20090232779 or US Patent Publication No.: 20090305405, the contents of each of which are herein incorporated in their entireties by reference. For example, cells can be differentiated to neural or glial lineages, using medium including any of the following factors in an effective combination: Brain derived neurotrophic factor (BDNF), neutrotrophin-3 (NT-3), NT-4, epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid, forskolin, fetal bovine serum (FBS), and bone morphogenic proteins (BMPs).

In other exemplary embodiments, cells with higher or increased developmental potential generated using the compositions and methods comprising synthetic, modified RNAs described herein can be differentiated into heptaocyte-like cells for treatment of liver diseases, such as cirrhosis. For example, cells can be differentiated to hepatocyte-like cells, using medium including any of the following factors in an effective combination or sequence: a hepatocyte supportive extracellular matrix, such as collagen or Matrigel; suitable differentiation agents, such as various isomers of butyrate and their analogs, exemplified by n-butyrate; a hepatocyte maturation factor, such as an organic solvent like dimethyl sulfoxide (DMSO); a maturation cofactor such as retinoic acid; a cytokine or hormone such as a glucocorticoid, epidermal growth factor (EGF), insulin, transforming growth factors (TGF-α and TGF-β), fibroblast growth factors (FGF), heparin, hepatocyte growth factors (HGF), interleukins (IL-1 and IL-6), insulin-like growth factors (IGF-I and IGF-II), and heparin-binding growth factors (HBGF-1).

The success of a differentiation program can be monitored by any of a number of criteria, including characterization of morphological features, detection or quantitation of expressed cell markers and enzymatic activity, and determination of the functional properties of the desired end cell types in vitro or in vivo. The level of mRNA corresponding to a marker can be determined both by in situ and by in vitro formats. The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. Protein markers can be measured e.g., by immunohistochemical techniques or the morphology of the cell can be monitored. Biochemical approaches, e.g., the ability of the differentiated cell to respond to a cell-type specific stimulus can also be monitored. An increase in the expression of a cell specific marker may be by about 5%, 10%, 25%, 50%, 75% or 100%. In one embodiment, the synthetic, modified RNA composition can direct cell fate towards different germ layers without definitively specifying a terminally differentiated cell type. For example, a synthetic, modified RNA encoding Sox17 or GATA6 can be used for definitive endodermal specification from pluripotent cells, such as an iPS or embryonic stem cell. Similarly, a synthetic, modified RNA encoding T (Brachyury) can be used for specification of mesoderm. For example, markers for neural cells include, but are not limited to: β-tubulin III or neurofilament, which are characteristic of neurons, glial fibrillary acidic protein (GFAP), present in astrocytes; galactocerebroside (GalC) or myelin basic protein (MBP), characteristic of oligodendrocytes; nestin, characteristic of neural precursors and other cells, and A2B5 and NCAM, characteristic of glial progenitors and neural progenitors, respectively. Similarly, an adipocyte can be detected by assaying for Oil-Red-O staining or acetylated LDL uptake. Cardiomyocytes can be detected by assaying for the expression of one or more cardiomyocyte specific markers, such as cardiotroponin I, Mef2c, connexin43, Nkx2.5, GATA-4, sarcomeric actinin, cariotroponin T and TBX5, and sarcomeric actinin, α-cardiac myosin heavy chain, actin, or ventricular myosin light chain 2 (MLC-2v). For skeletal muscle, markers include myoD, myogenin, and myf-5. Markers of interest for identifying liver cells include α-fetoprotein (liver progenitors); albumin, α₁-antitrypsin, glucose-6-phosphatase, cytochrome p450 activity, transferrin, asialoglycoprotein receptor, and glycogen storage (hepatocytes); CK7, CK19, and γ-glutamyl transferase (bile epithelium). The presence of endothelial cells can be detected by assaying the presence of an endothelial cell specific marker, such as CD31+, PECAM (platelet endothelial cell adhesion molecule), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. For pancreatic cells, pdx and insulin secretion can be used for determination of differentiation. The level of expression can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the markers; measuring the amount of protein encoded by the markers; or measuring the activity of the protein encoded by the markers.

In some embodiments, differentiation is detected by measuring an alteration in the morphology or biological function or activity of a differentiated cell. An alteration in biological function may be assayed, for example, by measuring an increase in acetylated LDL uptake in a reprogrammed adipocyte. For example, GABA-secreting neurons can be identified by production of glutamic acid decarboxylase or GABA. Dopaminergic neurons can be identified by production of dopa decarboxylase, dopamine, or tyrosine hydroxylase. Also, for example, differentiated hepatocyte lineage cells differentiated can be identified by α₁-antitrypsin (AAT) synthesis, albumin synthesis, evidence of glycogen storage, evidence of cytochrome p450 activity, and evidence of glucose-6-phosphatase activity. Other methods for assaying cell morphology and function are known in the art and are described in the Examples.

In some embodiments, the cells of the compositions and methods described herein are further cultured in the presence of cell specific growth factors, such as angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor-alpha, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2-alpha, cytokine-induced neutrophil chemotactic factor 2-beta, beta-endothelial cell growth factor, endothelia 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6 fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor b, fibroblast growth factor c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophil factor receptor-alpha-1, glial cell line-derived neutrophil factor receptor-alpha-2, growth related protein, growth related protein-alpha, growth related protein-beta, growth related protein-gamma, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor-alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor-alpha, platelet derived growth factor receptor-beta, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor-alpha, transforming growth factor-beta, transforming growth factor-beta-1, transforming growth factor-beta-1-2, transforming growth factor-beta-2, transforming growth factor-beta-3, transforming growth factor-beta-5, latent transforming growth factor-beta-1, transforming growth factor-beta-binding protein I, transforming growth factor-beta-binding protein II, transforming growth factor-beta-binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof. Such factors can also be injected or otherwise administered directly into an animal system for in vivo integration.

Cell Modifications

Homing Moieties and Cell-Surface Receptors

In some aspects and embodiments of the aspects described herein, a synthetic, modified RNA can be used to express a ligand or ligand receptor on the surface of a cell (e.g., a homing moiety). A ligand or ligand receptor moiety attached to a cell surface permits the cell to have a desired biological interaction with a tissue or an agent in vivo. A ligand can be an antibody, an antibody fragment, an aptamer, a peptide, a vitamin, a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surafce receptor, an adhesion molecule, a glycoprotein, a sugar residue, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof. For example, a ligand can be an antibody that recognizes a cancer-cell specific antigen, rendering the cell capable of preferentially interacting with tumor cells to permit tumor-specific localization of a modified cell. A ligand can confer the ability of a cell composition to accumulate in a tissue to be treated, since a preferred ligand is capable of interacting with a target molecule on the external face of a tissue to be treated. Ligands having limited cross-reactivity to other tissues are generally preferred.

In some cases, a ligand can act as a homing moiety which permits the cell to target to a specific tissue or interact with a specific ligand. Such homing moieties can include, for example, any member of a specific binding pair, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)₂ fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; and other homing moieties include for example, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety is a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of homing interactions.

A skilled artisan can select any homing moiety based on the desired localization or function of the cell, for example an estrogen receptor ligand, such as tamoxifen, can target cells to estrogen-dependent breast cancer cells that have an increased number of estrogen receptors on the cell surface. Other non-limiting examples of ligand/receptor interactions include CCR1 (e.g., for treatment of inflamed joint tissues or brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g., to target to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin), CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., for treatment of inflammation and inflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g., targeting to endothelium). In general, any receptor involved in targeting (e.g., cancer metastasis) can be harnessed for use in the methods and compositions described herein. Table 2 and Table 3 provide non-limiting examples of CD (“cluster of differentiation”) molecules and other cell-surface/membrane bound molecules and receptors that can be expressed using the synthetic, modified RNA compositions and methods described herein for targeting and homing to cells of interest, or for changing the phenotype of a cell.

Mediators of Cell Death

In one embodiment, a synthetic, modified RNA composition can be used to induce apoptosis in a cell (e.g., a cancer cell) by increasing the expression of a death receptor, a death receptor ligand or a combination thereof. This method can be used to induce cell death in any desired cell and has particular usefulness in the treatment of cancer where cells escape natural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways that converge upon a final effector mechanism consisting of multiple interactions between several “death receptors” and their ligands, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily. The best-characterized death receptors are CD95 (“Fas”), TNFR1 (p55), death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). The final effector mechanism of apoptosis is the activation of a series of proteinases designated as caspases. The activation of these caspases results in the cleavage of a series of vital cellular proteins and cell death. The molecular mechanism of death receptors/ligands-induced apoptosis is well known in the art. For example, Fas/FasL-mediated apoptosis is induced by binding of three FasL molecules which induces trimerization of Fas receptor via C-terminus death domains (DDs), which in turn recruit an adapter protein FADD (Fas-associated protein with death domain) and Caspase-8. The oligomerization of this trimolecular complex, Fas/FAIDD/caspase-8, results in proteolytic cleavage of proenzyme caspase-8 into active caspase-8 that, in turn, initiates the apoptosis process by activating other downstream caspases through proteolysis, including caspase-3. Death ligands in general are apoptotic when formed into trimers or higher order of structures. As monomers, they may serve as antiapoptotic agents by competing with the trimers for binding to the death receptors.

In one embodiment, the synthetic, modified RNA composition encodes for a death receptor (e.g., Fas, TRAIL, TRAMO, TNFR, TLR etc). Cells made to express a death receptor by transfection of modified RNA become susceptible to death induced by the ligand that activates that receptor. Similarly, cells made to express a death ligand, e.g., on their surface, will induce death of cells with the receptor when the transfected cell contacts the target cell. In another embodiment, the modified RNA composition encodes for a death receptor ligand (e.g., FasL, TNF, etc). In another embodiment, the modified RNA composition encodes a caspase (e.g., caspase 3, caspase 8, caspase 9 etc). Where cancer cells often exhibit a failure to properly differentiate to a non-proliferative or controlled proliferative form, in another embodiment, the synthetic, modified RNA composition encodes for both a death receptor and its appropriate activating ligand. In another embodiment, the synthetic, modified RNA composition encodes for a differentiation factor that when expressed in the cancer cell, such as a cancer stem cell, will induce the cell to differentiate to a non-pathogenic or non-self-renewing phenotype (e.g., reduced cell growth rate, reduced cell division etc) or to induce the cell to enter a dormant cell phase (e.g., Go resting phase).

One of skill in the art will appreciate that the use of apoptosis-inducing techniques will require that the synthetic, modified RNAs are appropriately targeted to e.g., tumor cells to prevent unwanted wide-spread cell death. Thus, one can use a delivery mechanism (e.g., attached ligand or antibody, targeted liposome etc) that recognizes a cancer antigen such that the modified RNAs are expressed only in cancer cells.

Cellular Therapies and Cellular Administration

The compositions and methods comprising synthetic, modified RNAs are particularly useful for generating cells, such as differentiated cells, for use in patients in need of cellular therapies or regenerative medicine applications. Accordingly, various embodiments of the methods and compositions described herein involve administration of an effective amount of a cell or a population of cells, generated using any of the compositions or methods comprising synthetic, modified RNAs described herein, to an individual or subject in need of a cellular therapy. The cell or population of cells being administered can be an autologous population, or be derived from one or more heterologous sources. The cell can be, for example, a stem cell, such as a lineage-restricted progenitor cell, multipotent cell, or an oligopotent cell, or a fully or partially differentiated progeny of a stem cell. In some embodiments, the stem cell can be generated through the introduction of synthetic, modified RNAs encoding differentiation factor(s) as described herein. In addition, the population of cells administered can be of a lineage selected from one of an ecotodermal lineage, a mesodermal lineage, or an endodermal lineage. The cell can also be a cell modified to express a targeting moiety or a mediator of targeted cell death, using synthetic, modified RNAs as described herein. Further, such differentiated cells can be administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area. In some such embodiments, differentiated cells can be introduced to a scaffold or other structure to generate, for example, a tissue ex vivo, that can then be introduced to a patient. For example, islet precursor cells or their derivatives can be generated to restore islet function in a patient having any condition relating to inadequate production of a pancreatic endocrine (insulin, glucagon, or somatostatin), or the inability to properly regulate secretion, e.g., Type I (insulin-dependent) diabetes mellitus.

A variety of means for administering cells to subjects are known to those of skill in the art. Such methods can include systemic injection, for example i.v. injection, or implantation of cells into a target site in a subject. Cells may be inserted into a delivery device which facilitates introduction by injection or implantation into the subject. Such delivery devices can include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In one preferred embodiment, the tubes additionally have a needle, e.g., through which the cells can be introduced into the subject at a desired location. The cells can be prepared for delivery in a variety of different forms. For example, the cells can be suspended in a solution or gel or embedded in a support matrix when contained in such a delivery device. Cells can be mixed with a pharmaceutically acceptable carrier or diluent in which the cells remain viable.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid. Preferably, prior to the introduction of cells as described herein, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

It is preferred that the mode of cell administration is relatively non-invasive, for example by intravenous injection, pulmonary delivery through inhalation, topical, or intranasal administration. However, the route of cell administration will depend on the tissue to be treated and may include implantation. Methods for cell delivery are known to those of skill in the art and can be extrapolated by one skilled in the art of medicine for use with the methods and compositions described herein.

Direct injection techniques for cell administration can also be used to stimulate transmigration of cells through the entire vasculature, or to the vasculature of a particular organ, such as for example liver, or kidney or any other organ. This includes non-specific targeting of the vasculature. One can target any organ by selecting a specific injection site, e.g., a liver portal vein. Alternatively, the injection can be performed systemically into any vein in the body. This method is useful for enhancing stem cell numbers in aging patients. In addition, the cells can function to populate vacant stem cell niches or create new stem cells to replenish the organ, thus improving organ function. For example, cells may take up pericyte locations within the vasculature. In another example, neural stem cells or precursor cells generated using the compositions and methods comprising synthetic, modified RNAs are transplanted directly into parenchymal or intrathecal sites of the central nervous system, according to the disease being treated, such as for example, a spinal cord injury. Grafts can be done using single cell suspension or small aggregates at a density of 25,000-500,000 cells per mL (U.S. Pat. No. 5,968,829, the contents of which are herein incorporated in their entireties by reference). A successful transplant can show, for example, transplant-derived cells present in the lesion 2-5 weeks later, differentiated into astrocytes, oligodendrocytes, and/or neurons, and migrating along the cord from the lesioned end.

If so desired, a mammal or subject can be pre-treated with an agent, for example an agent is administered to enhance cell targeting to a tissue (e.g., a homing factor) and can be placed at that site to encourage cells to target the desired tissue. For example, direct injection of homing factors into a tissue can be performed prior to systemic delivery of ligand-targeted cells.

Scaffolds and Tissue Engineering

It is further contemplated that, in some embodiments of these aspects, cells generated by differentiation or transdifferentiation using the synthetic, modified RNAs described herein, can not only be administered as cells in suspension, but also as cells populating a matrix, scaffold, or other support to create an artificial tissue, for use in cellular therapies in regenerative medicine and tissue engineering.

Tissue engineering refers to the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors for the de novo generation of tissue or tissue structures. Such engineered tissue or tissue structures are useful for therapeutic purposes to improve or replace biological functions. As used herein, “engineered tissue” encompasses a broad range of applications, including, but not limited to, utility in the repair or replace portions of, or whole tissues (e.g., heart, cardiac tissue, ventricular myocardium, and other tissues such as bone, cartilage, pancreas, liver, kidney, blood vessels, bladder, etc.), or in assays for identifying agents which modify the function of parts of, or entire organs without the need to obtain such organs from a subject.

In some embodiments, a “support” i.e., any suitable carrier material to which cells generated using the methods and compositions comprising synthetic, modified RNAs described herein are able to attach themselves or adhere, is used in order to form a corresponding cell composite, e.g. an artificial tissue. In some embodiments, a matrix or carrier material, respectively, is present already in a three-dimensional form desired for later application. For example, bovine pericardial tissue can be used as matrix which is crosslinked with collagen, decellularized and photofixed.

In some such embodiments, a scaffold, which can also be referred to as a “biocompatible substrate,” can be used as a material that is suitable for implantation into a subject onto which a cell population can be deposited. A biocompatible substrate does not cause toxic or injurious effects once implanted in the subject. In one embodiment, the biocompatible substrate is a polymer with a surface that can be shaped into a desired structure that requires repairing or replacing. The polymer can also be shaped into a part of a structure that requires repairing or replacing. The biocompatible substrate provides the supportive framework that allows cells to attach to it, and grow on it. Cultured populations of cells can then be grown on the biocompatible substrate, which provides the appropriate interstitial distances required for cell-cell interaction.

A structure or scaffold can be used to aid in further controlling and directing a cell or population of cells undergoing differentiation or transdifferentiation using the compositions and methods described herein. A structure or scaffold, such as a biopolymer structure, can be designed to provide environmental cues to control and direct the differentiation of cells to a functionally active engineered tissue, e.g., multipotent cells undergoing differentiation, using the synthetic, modified RNAs described herein, into ventricular cardiomyocytes to generate a functional, contracting tissue myocardium structure. By “functionally active,” it is meant that the cell attached to the scaffold comprises at least one function of that cell type in its native environment. A structure or scaffold can be engineered from a nanometer to micrometer to millimeter to macroscopic length, and can further comprise or be based on factors such as, but not limited to, material mechanical properties, material solubility, spatial patterning of bioactive compounds, spatial patterning of topological features, soluble bioactive compounds, mechanical perturbation (cyclical or static strain, stress, shear, etc. . . . ), electrical stimulation, and thermal perturbation.

The construction of an engineered tissue can be carried out by first assembling the scaffolds, and then seeding with a cell type that has undergone differentiation or partial differentiation using the synthetic, modified RNA compositions and methods described herein. Alternatively, an engineered tissue can be made by seeding a matrix or other scaffold component cell with cells, such as iPS cells or human ES cells, and applying or introducing a desired synthetic, modified RNA composition directly to the scaffold comprising the cells. A scaffold can be in any desired geometric conformation, for example, a flat sheet, a spiral, a cone, a v-like structure and the like. A scaffold can be shaped into, e.g., a heart valve, vessel (tubular), planar construct or any other suitable shape. Such scaffold constructs are known in the art (see, e.g., WO02/035992, U.S. Pat. Nos. 6,479,064, 6,461,628, the contents of which are herein incorporated in their entireties by reference). In some embodiments, after culturing the cells on the scaffold, the scaffold is removed (e.g., bioabsorbed or physically removed), and the layers of differentiation or transdifferentiated cells maintain substantially the same conformation as the scaffold, such that, for example, if the scaffold was spiral shaped, the cells form a 3D-engineered tissue that is spiral shaped. In addition, it is contemplated that different synthetic, modified RNA compositions can be contacted with or applied to a scaffold comprising cells in order to allow the growth and differentiation of a plurality of different, differentiated cells types to form a desired engineered tissue. For example, for construction of muscle tissue with blood vessels, a scaffold can be seeded with different population of cells which make up blood vessels, neural tissue, cartilage, tendons, ligaments and the like.

Biopolymer structures can be generated by providing a transitional polymer on a substrate; depositing a biopolymer on the transitional polymer; shaping the biopolymer into a structure having a selected pattern on the transitional polymer (poly(N-Isopropylacrylamide); and releasing the biopolymer from the transitional polymer with the biopolymer's structure and integrity intact. A biopolymer can be selected from an extracellular matrix (ECM) protein, growth factor, lipid, fatty acid, steroid, sugar and other biologically active carbohydrates, a biologically derived homopolymer, nucleic acids, hormone, enzyme, pharmaceutical composition, cell surface ligand and receptor, cytoskeletal filament, motor protein, silks, polyprotein (e.g., poly(lysine)) or any combination thereof. The biopolymers used in the generation of the scaffolds for the embodiments directed to tissue engineering described herein include, but are not limited to, a) extracellular matrix proteins to direct cell adhesion and function (e.g., collagen, fibronectin, laminin, etc.); (b) growth factors to direct cell function specific to cell type (e.g., nerve growth factor, bone morphogenic proteins, vascular endothelial growth factor, etc.); (c) lipids, fatty acids and steroids (e.g., glycerides, non-glycerides, saturated and unsaturated fatty acids, cholesterol, corticosteroids, sex steroids, etc.); (d) sugars and other biologically active carbohydrates (e.g., monosaccharides, oligosaccharides, sucrose, glucose, glycogen, etc.); (e) combinations of carbohydrates, lipids and/or proteins, such as proteoglycans (protein cores with attached side chains of chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, and/or keratan sulfate); glycoproteins [e.g., selectins, immunoglobulins, hormones such as human chorionic gonadotropin, Alpha-fetoprotein and Erythropoietin (EPO), etc.]; proteolipids (e.g., N-myristoylated, palmitoylated and prenylated proteins); and glycolipids (e.g., glycoglycerolipids, glycosphingolipids, glycophosphatidylinositols, etc.); (f) biologically derived homopolymers, such as polylactic and polyglycolic acids and poly-L-lysine; (g) nucleic acids (e.g., DNA, RNA, etc.); (h) hormones (e.g., anabolic steroids, sex hormones, insulin, angiotensin, etc.); (i) enzymes (types: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases; examples: trypsin, collegenases, matrix metallproteinases, etc.); (j) pharmaceuticals (e.g., beta blockers, vasodilators, vasoconstrictors, pain relievers, gene therapy, viral vectors, anti-inflammatories, etc.); (k) cell surface ligands and receptors (e.g., integrins, selectins, cadherins, etc.); (l) cytoskeletal filaments and/or motor proteins (e.g., intermediate filaments, microtubules, actin filaments, dynein, kinesin, myosin, etc.), or any combination thereof. For example, a biopolymer can be selected from the group consisting of fibronectin, vitronectin, laminin, collagen, fibrinogen, silk or silk fibroin.

Following or during construction of a biopolymer scaffold, cells can be integrated into or onto the scaffold. In some embodiments, the cells to be differentiated are human ES-derived cells or iPS-derived cells, and the methods further comprise growing the cells in the scaffold where the structure, composition, ECM type, growth factors and/or other cell types can assist in differentiation of the cells into the desired differentiated cell type. In some embodiments, such engineered tissue can be further used in drug screening applications. For example, an engineered myocardium tissue composition can be useful as a tool to identify agents which modify the function of cardiac muscle (e.g., to identify cardiotoxic agents).

Other exemplary materials suitable for polymer scaffold fabrication include, but are not limited to, polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, Teflon™, nylon silicon, and shape memory materials, such as poly(styrene-block-butadiene), polynorbornene, hydrogels, metallic alloys, and oligo(ε-caprolactone)diol as switching segment/oligo(p-dioxyanone)diol as physical crosslink. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989), the contents of which are herein incorporated in their reference by entirety.

In some embodiments, additional bioactive substances can be added to a biopolymer scaffold comprising cells being differentiated using the synthetic, modified RNA compositions described herein, such as, but not limited to, demineralized bone powder as described in U.S. Pat. No. 5,073,373 the contents of which are incorporated herein by reference; collagen, insoluble collagen derivatives, etc., and soluble solids and/or liquids dissolved therein; antiviricides, particularly those effective against HIV and hepatitis; antimicrobials and/or antibiotics such as erythromycin, bacitracin, neomycin, penicillin, polymycin B, tetracyclines, biomycin, chloromycetin, and streptomycins, cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc.; biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids; peptides; vitamins; inorganic elements; co-factors for protein synthesis; hormones; endocrine tissue or tissue fragments; synthesizers; enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases, etc.; polymer cell scaffolds with parenchymal cells; angiogenic agents and polymeric carriers containing such agents; collagen lattices; antigenic agents; cytoskeletal agents; cartilage fragments; living cells such as chondrocytes, bone marrow cells, mesenchymal stem cells; natural extracts; genetically engineered living cells or otherwise modified living cells; expanded or cultured cells; DNA delivered by plasmid, viral vectors or other means; tissue transplants; demineralized bone powder; autogenous tissues such as blood, serum, soft tissue, bone marrow, etc.; bioadhesives; bone morphogenic proteins (BMPs); osteoinductive factor (IFO); fibronectin (FN); endothelial cell growth factor (ECGF); vascular endothelial growth factor (VEGF); cementum attachment extracts (CAE); ketanserin; human growth hormone (HGH); animal growth hormones; epidermal growth factor (EGF); interleukins, e.g., interleukin-1 (IL-1), interleukin-2 (IL-2); human alpha thrombin; transforming growth factor (TGF-beta); insulin-like growth factors (IGF-1, IGF-2); platelet derived growth factors (PDGF); fibroblast growth factors (FGF, BFGF, etc.); periodontal ligament chemotactic factor (PDLGF); enamel matrix proteins; growth and differentiation factors (GDF); hedgehog family of proteins; protein receptor molecules; small peptides derived from growth factors above; bone promoters; cytokines; somatotropin; bone digestors; antitumor agents; cellular attractants and attachment agents; immuno-suppressants; permeation enhancers, e.g., fatty acid esters such as laureate, myristate and stearate monoesters of polyethylene glycol, enamine derivatives, alpha-keto aldehydes, etc.; and nucleic acids. The amounts of such optionally added bioactive substances can vary widely with optimum levels being readily determined in a specific case by routine experimentation.

Diseases Treatable by Cell Transplantation

A wide range of diseases are recognized as being treatable with cellular therapies. Accordingly, also provided herein are compositions and methods comprising synthetic, modified RNAs for generating cells for use in cellular therapies, such as stem cell therapies. As non-limiting examples, these include diseases marked by a failure of naturally occurring stem cells, such as aplastic anemia, Fanconi anemia, and paroxysmal nocturnal hemoglobinuria (PNH). Others include, for example: acute leukemias, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute biphenotypic leukemia and acute undifferentiated leukemia; chronic leukemias, including chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile chronic myelogenous leukemia (JCML) and juvenile myelomonocytic leukemia (JMML); myeloproliferative disorders, including acute myelofibrosis, angiogenic myeloid metaplasia (myelofibrosis), polycythemia vera and essential thrombocythemia; lysosomal storage diseases, including mucopolysaccharidoses (MPS), Hurler's syndrome (MPS-IH), Scheie syndrome (MPS-IS), Hunter's syndrome (MPS-II), Sanfilippo syndrome (MPS-III), Morquio syndrome (MPS-IV), Maroteaux-Lamy Syndrome (MPS-VI), Sly syndrome, beta-glucuronidase deficiency (MPS-VII), adrenoleukodystrophy, mucolipidosis II (I-cell Disease), Krabbe disease, Gaucher's disease, Niemann-Pick disease, Wolman disease and metachromatic leukodystrophy; histiocytic disorders, including familial erythrophagocytic lymphohistiocytosis, histiocytosis-X and hemophagocytosis; phagocyte disorders, including Chediak-Higashi syndrome, chronic granulomatous disease, neutrophil actin deficiency and reticular dysgenesis; inherited platelet abnormalities, including amegakaryocytosis/congenital thrombocytopenia; plasma cell disorders, including multiple myeloma, plasma cell leukemia, and Waldenstrom's macroglobulinemia. Other malignancies treatable with stem cell therapies include but are not limited to breast cancer, Ewing sarcoma, neuroblastoma and renal cell carcinoma, among others. Also treatable with stem cell therapy are: lung disorders, including COPD and bronchial asthma; congenital immune disorders, including ataxia-telangiectasia, Kostmann syndrome, leukocyte adhesion deficiency, DiGeorge syndrome, bare lymphocyte syndrome, Omenn's syndrome, severe combined immunodeficiency (SCID), SCID with adenosine deaminase deficiency, absence of T & B cells SCID, absence of T cells, normal B cell SCID, common variable immunodeficiency and X-linked lymphoproliferative disorder; other inherited disorders, including Lesch-Nyhan syndrome, cartilage-hair hypoplasia, Glanzmann thrombasthenia, and osteopetrosis; neurological conditions, including acute and chronic stroke, traumatic brain injury, cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis and epilepsy; cardiac conditions, including atherosclerosis, congestive heart failure and myocardial infarction; metabolic disorders, including diabetes; and ocular disorders including macular degeneration and optic atrophy. Such diseases or disorders can be treated either by administration of stem cells themselves, permitting in vivo differentiation to the desired cell type with or without the administration of agents to promote the desired differentiation, or by administering stem cells differentiated to the desired cell type in vitro. Efficacy of treatment is determined by a statistically significant change in one or more indicia of the targeted disease or disorder.

Dosage and Administration

Dosage and administration will vary with the condition to be treated and the therapeutic approach taken in a given instance.

Depending on the disease or disorder being treated and on the approach being taken, cells over a range of, for example, 2-5×10⁵, or more, e.g., 1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or more can be administered. Where differentiated cells are to be administered, the dose will most often be higher than where stem cells are administered, because differentiated cells will have reduced or limited capacity for self-renewal compared to stem cells. Repeat administration of differentiated cells may be necessary if the cells are not capable of self-renewal.

It is contemplated that cells generated by differentiation or transdifferentiation can be administered as cells in suspension, or as cells populating a matrix, scaffold, or other support to create an artificial tissue. To this end, resorbable matrices and scaffolds are known in the art, as are approaches for populating them with cells, as has been described herein. As but one example, matrices fabricated out of silk proteins are well suited as supports for cells, and are known to be well tolerated for implantation. Cells as described herein can be seeded on such matrices either alone or in combination with other cells, including autologous cells from the intended recipient, to provide the necessary environment for growth and maintenance of the cells in the desired differentiated (or non-differentiated) state. It is also contemplated that the cells generated by differentiation or transdifferentiation can be administered to a subject in need thereof, in an encapsulated form, according to known encapsulation technologies, including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350, which are incorporated herein in their entireties by reference). Where the differentiated or transdifferentiated cells are encapsulated, in some embodiments the cells are encapsulated by macroencapsulation, as described in U.S. Pat. Nos. 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and published PCT patent application WO 95/05452, which are incorporated herein in their entireties by reference. In such embodiments, cells on the order of 1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or more can be administered alone or on a matrix or support.

In other embodiments, cells can be suspended in a gel for administration to keep them relatively localized.

The success of treatment can be evaluated by the ordinarily skilled clinician by monitoring one or more symptoms or markers of the disease or disorder being treated by administration of the cells. Effective treatment includes any statistically significant improvement in one or more indicia of the disease or disorder. Where appropriate, a clinically accepted grade or scaling system for the given disease or disorder can be applied, with an improvement in the scale or grade being indicative of effective treatment.

In those aspects and embodiments where synthetic, modified RNAs are to be administered directly, instead of cells treated with or resulting from treatment with synthetic, modified RNA, the dosages will also vary depending upon the approach taken, the mode of delivery and the disease to be treated. For example, systemic administration without a targeting approach will generally require greater amounts of synthetic, modified RNA than either local administration or administration that employs a targeting or homing approach. Depending upon the targeted cell or tissue and the mode of delivery, effective dosages of synthetic, modified RNA can include, for example, 1 ng/kg of body weight up to a gram or more per kg of body weight and any amount in between. Preferred amounts can be, for example, in the range of 5 μg/kg body weight to 30 μg/kg of body weight or any amount in between. Dosages in such ranges can be administered once, twice, three times, four times or more per day, or every two days, every three days, every four days, once a week, twice a month, once a month or less frequently over a duration of days, weeks or months, depending on the condition being treated—where the therapeutic approach treats or ameliorates but does not permanently cure the disease or disorder, e.g., where the synthetic, modified RNA effects treatment of a metabolic disorder by expression of a protein that is deficient in the subject, administration of modified RNA can be repeated over time as needed. Where, instead, the synthetic, modified RNA leads to the establishment of a cell compartment that maintains itself and treats the disease or disorder, readministration may become unnecessary. Sustained release formulations of synthetic, modified RNA compositions are specifically contemplated herein. Continuous, relatively low doses are contemplated after an initial higher therapeutic dose.

A pharmaceutical composition that includes at least one synthetic, modified RNA described herein can be delivered to or administered to a subject by a variety of routes depending upon whether local or systemic treatment is desired and upon the area to be treated. Exemplary routes include parenteral, intrathecal, parenchymal, intravenous, nasal, oral, and ocular delivery routes. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. A synthetic, modified RNA can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more synthetic, modified RNAs and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into the compositions. Compositions for intrathecal or intraventricular administration of synthetic, modified RNAs can include sterile aqueous solutions that can also contain buffers, diluents and other suitable additives.

In some embodiments, the effective dose of a synthetic, modified RNA can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, a non-implantable delivery device, e.g., needle, syringe, pen device, or implantatable delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir can be advisable. In some such embodiments, the delivery device can include a mechanism to dispense a unit dose of the pharmaceutical composition comprising a synthetic, modified RNA. In some embodiments, the device releases the pharmaceutical composition comprising a synthetic, modified RNA continuously, e.g., by diffusion. In some embodiments, the device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, “topical delivery” can refer to the direct application of a synthetic, modified RNA to any surface of the body, including the eye, a mucous membrane, surfaces of a body cavity, or to any internal surface. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Topical administration can also be used as a means to selectively deliver the synthetic, modified RNA to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

Formulations for parenteral administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

A synthetic, modified RNA can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition comprising a synthetic, modified RNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into the lung cells to directly transfect the lung cells, and/or enter the blood circulation. Direct transfection by inhalation will allow expression of a desired protein, for example CFTR, by the transfected lung cells. Accordingly, pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs. Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations of the compositions comprising synthetic, modified RNAs described herein. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that can be readily formulated as dry powders. A synthetic, modified RNA composition can be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition comprising a synthetic, modified RNA for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

A synthetic, modified RNA can be modified such that it is capable of traversing the blood brain barrier. For example, the synthetic, modified RNA can be conjugated to a molecule that enables the agent to traverse the barrier. Such conjugated synthetic, modified RNA can be administered by any desired method, such as by intraventricular or intramuscular injection, or by pulmonary delivery, for example.

A composition comprising a synthetic, modified RNA described herein can also be delivered through the use of implanted, indwelling catheters that provide a means for injecting small volumes of fluid containing the synthetic, modified RNAs described herein directly into local tissues. The proximal end of these catheters can be connected to an implanted, access port surgically affixed to the patient's body, or to an implanted drug pump located in, for example, the patient's torso.

Alternatively, implantable delivery devices, such as an implantable pump can be employed. Examples of the delivery devices for use with the compositions comprising a synthetic, modified RNA described herein include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously in the body or on the cranium, and provides an access port through which therapeutic agents can be delivered. In addition to the aforementioned device, the delivery of the compositions comprising a synthetic, modified RNA described herein can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference. Using the teachings described herein, those of skill in the art will recognize that these and other devices and systems can be suitable for delivery of compositions comprising the synthetic, modified RNAs described herein.

In some such embodiments, the delivery system further comprises implanting a pump outside the body, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of a composition comprising a synthetic, modified RNA described herein through the discharge portion of the catheter. A further embodiment comprises periodically refreshing a supply of the composition comprising a synthetic, modified RNA to the pump outside the body.

A synthetic, modified RNA can be administered ocularly, such as to treat retinal disorders, e.g., a retinopathy. For example, the pharmaceutical compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Ointments or droppable liquids can be delivered by ocular delivery systems known in the art, such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The pharmaceutical composition can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. The composition containing the synthetic, modified RNA can also be applied via an ocular patch.

A synthetic, modified RNA can be administered by an oral or nasal delivery. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.

Administration of a composition comprising a synthetic, modified RNA can be provided by the subject or by another person, e.g., a another caregiver. A caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.

Where cells expressing proteins encoded by synthetic, modified RNA as described herein are administered to treat a malignancy or disease or disorder, the dose of cells administered will also vary with the therapeutic approach. For example, where the cell expresses a death ligand targeting the tumor cell, the dosage of cells administered will vary with the mode of their administration, e.g., local or systemic (smaller doses are required for local), and with the size of the tumor being treated—generally more cells or more frequent administration is warranted for larger tumors versus smaller ones. The amount of cells administered will also vary with the level of expression of the polypeptide or polypeptides encoded by the modified RNA—this is equally true of the administration of cells expressing proteins encoded by modified RNA for any purpose described herein. An important advantage of the methods described herein is that where, for example, more than one factor or polypeptide is expressed from a modified RNA introduced to a cell, the relative dosage of the expressed proteins can be tuned in a straightforward manner by adjusting the relative amounts of the modified RNAs introduced to the cell or subject. This is in contrast to the difficulty of tuning the expression of even a single gene product in a cell transduced with a viral or even a plasmid vector.

Therapeutic compositions containing at least one synthetic, modified-NA can be conventionally administered in a unit dose. The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired.

Pharmaceutical Compositions

The present invention involves therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions contain a physiologically tolerable carrier together with an active compound (synthetic, modified RNA, a cell transfected with a synthetic, modified RNA, or a cell differentiated, de-differentiated or transdifferentiated with a synthetic, modified RNA) as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired. As used herein, the terms “pharmaceutically acceptable,” “physiologically tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable or unacceptable physiological effects such as toxicity, nausea, dizziness, gastric upset, immune reaction and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectable either as liquid solutions or suspensions, however, particularly where synthetic, modified RNA itself is administered, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Saline-based carriers are most useful for the administration of cells or cell preparations. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.

Kits

Provided herein are kits comprising synthetic, modified RNAs as described herein and kits for preparing such synthetic, modified RNAs.

Provided herein, in some aspects, are kits for altering the phenotype or the developmental potential of a cell, and comprise (a) a synthetic, modified RNA composition comprising at least one synthetic, modified RNA molecule comprising: (i) a 5′ cap, (ii) an open reading frame encoding a polypeptide, and (iii) at least one modified nucleoside, and (b) packaging and instructions therefor.

In one embodiment of this aspect, the synthetic, modified RNA composition can further comprise a 3′ untranslated region (e.g., murine alpha-globin 3′ untranslated region) to enhance the stability of the synthetic, modified RNA. In another embodiment of this aspect, the 5′ cap is a 5′ cap analog such as e.g., a 5′ diguanosine cap, tetraphosphate cap analogs having a methylene-bis(phosphonate) moiety (see e.g., Rydzik, A M et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA 14(6):1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g., Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien, E. et al., (2004) RNA 10(9):1479-1487), or anti-reverse cap analogs (see e.g., Jemielity, J. et al., (2003) RNA 9(9): 1108-1122 and Stepinski, J. et al., (2001) RNA 7(10): 1486-1495).

In other embodiments, the kit can further comprise materials for further reducing the innate immune response of a cell. For example, the kit can further comprise a soluble interferon receptor, such as B18R. The synthetic, modified RNAs provided in such a kit can encode for a polypeptide to express a transcription factor, a targeting moiety, a cell type-specific polypeptide, a cell-surface polypeptide, a differentiation factor, a reprogramming factor or a de-differentiation factor. The synthetic, modified RNA can be provided such that the synthetic, modified RNA is dephosphorylated, lacks a 5′ phosphate, comprises a 5′ monophosphate, or lacks a 5′ triphosphate.

In some embodiments, the kit can comprise a plurality of different synthetic, modified RNA molecules.

In some aspects, the kit can be provided to induce reprogramming of a somatic cell to an induced pluripotent stem cell. Such kits include synthetic, modified RNAs encoding Oct4, Klf4, Sox2, or MYC. In some embodiments, the kits further comprise a synthetic, modified RNAs encoding LIN-28. The kit can provide the synthetic, modified RNAs in an admixture or as separate RNA aliquots.

The kit can further comprise an agent to enhance efficiency of reprogramming (e.g., valproic acid). The kit can further comprise one or more antibodies or primer reagents to detect a cell-type specific marker to identify reprogrammed cells.

Also provided herein are kits for preparing a synthetic, modified RNA. The kit comprises at least one modified nucleoside, such as 5′-methylcytidine or pseudouridine and an RNA polymerase. The kit can also comprise a 5′ cap analog. The kit can also comprise a phosphatase enzyme (e.g., Calf intestinal phosphatase) to remove the 5′ triphosphate during the RNA modification procedure. The kit can also comprise one or more templates for the generation of a synthetic, modified-RNA.

In one aspect, provided herein are kits comprising: (a) a container or vial with at least one synthetic, modified RNA molecule comprising at least two modified nucleosides, and (b) packaging and instructions therefor. Optionally, the kit can comprise one or more control synthetic, modified RNAs, such as a synthetic, modified RNA encoding green fluorescent protein (GFP) or other marker molecule. In some embodiments of this aspect, the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I). In some embodiments of this aspect, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect, the container with at least one synthetic, modified RNA molecule comprising at least two modified nucleosides further comprises a buffer. In some such embodiments, the buffer is RNase-free TE buffer at pH 7.0. In some embodiments of this aspect, the kit further comprises a container with cell culture medium.

In some embodiments of this aspect, the at least one synthetic, modified RNA encodes a developmental potential altering factor. In some such embodiments, the developmental potential altering factor is a reprogramming factor, a differentiation factor, or a de-differentiation factor.

In some embodiments of this aspect, the kit further comprises a container or vial comprising IFN inhibitor. In some embodiments of this aspect, the kit further comprises a container or vial valproic acid.

In some embodiments of this aspect, the synthetic, modified RNA encoding a reprogramming factor in the vial or container has a concentration of 100 ng/μl.

In some embodiments of this aspect, the reprogramming factor is selected from the group consisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some such embodiments, the kit comprises at least three of the reprogramming factors selected from the group consisting of OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28. In some embodiments, the kit does not comprise a synthetic, modified RNA encoding c-MYC.

In some embodiments of those aspects where the kit is provided to induce reprogramming of a somatic cell to an induced pluripotent stem cell, the kit comprises: a vial comprising a synthetic, modified RNA encoding OCT4 and a buffer; a vial comprising a synthetic, modified RNA encoding SOX2 and a buffer; a vial comprising a synthetic, modified RNA encoding c-MYC and a buffer; and a vial comprising a synthetic, modified RNA encoding KLF4 and a buffer. In some such embodiments, the concentration of each reprogramming factor in the vial is 100 ng/μl. In some embodiments, the at least two modified nucleosides are pseudouridine and 5-methylcytodine. In some embodiments, OCT4 is provided in the kit in a molar excess of about three times the concentration of KLF4, SOX-2, and c-MYC in the kit. In some such embodiments, the kit further comprises a vial comprising a synthetic, modified RNA molecule encoding LIN28 and a buffer. In some such embodiments, the buffer is RNase-free TE buffer at pH 7.0. In some embodiments, the kit further comprises a synthetic, modified RNA encoding a positive control molecule, such as GFP.

For example, in one embodiment of those aspects where the kit is provided to induce reprogramming of a somatic cell to an induced pluripotent stem cell, the kit comprises: a vial comprising a synthetic, modified RNA encoding OCT4 and a buffer; a vial comprising a synthetic, modified RNA encoding SOX2 and a buffer; a vial comprising a synthetic, modified RNA encoding c-MYC and a buffer; a vial comprising a synthetic, modified RNA encoding KLF4 and a buffer; a vial comprising a synthetic, modified RNA molecule encoding LIN28 and a buffer; a vial comprising a synthetic, modified RNA encoding a positive control GFP molecule; and packaging and instructions therefor; where the concentration of the synthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 and GFP in each of the said vials is 100 ng/μl, wherein the buffers in each of said vials is RNase-free TE buffer at pH 7.0; and wherein the synthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 and GFP all comprise pseudouridine and 5-methylcytidine nucleoside modifications.

In other embodiments of those aspects where the kit is provided to induce reprogramming of a somatic cell to an induced pluripotent stem cell, the kit comprises: a single container or vial comprising all the synthetic, modified RNAs provided in the kit. In some such embodiments, the kit comprises a single vial or single container comprising: a synthetic, modified RNA encoding OCT4; a synthetic, modified RNA encoding SOX2; a synthetic, modified RNA encoding c-MYC; a synthetic, modified RNA encoding KLF4; and a buffer. In some such embodiments, the buffer is RNase-free TE buffer at pH 7.0. In some such embodiments, the total concentration of reprogramming factors in the vial is 100 ng/μl. In some embodiments, the at least two modified nucleosides are pseudouridine and 5-methylcytodine. In some such embodiments, OCT4 is provided in the vial or container in a molar excess of about three times the concentration of KLF4, SOX-2, and c-MYC in the vial or container. In some such embodiments, the vial or container further comprises a synthetic, modified RNA molecule encoding LIN28. In some such embodiments, the buffer is RNase-free TE buffer at pH 7.0. In some embodiments, the kit further comprises a synthetic, modified RNA encoding a positive control molecule, such as GFP.

In some embodiments, the kits provided herein comprise at least one synthetic, modified RNA further comprising a 5′ cap. In some such embodiments, the 5′ cap is a 5′ cap analog. In some such embodiments, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments, t the kits provided herein comprise at least one synthetic, modified RNA that does not comprise a 5′ triphosphate.

In some embodiments, the kits provided herein comprise at least one synthetic and modified RNA further comprising a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated regions, or any combination thereof. In some such embodiments, the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof, comprises one or more modified nucleosides.

All kits described herein can further comprise a buffer, a cell culture medium, a transfection medium and/or a media supplement. In preffered embodiments, the buffers, cell culture mediums, transfection mediums, and/or media supplements are RNase-free. In some embodiments, the synthetic, modified RNAs provided in the kits can be in a non-solution form of specific quantity or mass, e.g., 20 μg, such as a lyophilized powder form, such that the end-user adds a suitable amount of buffer or medium to bring the synthetic, modified RNAs to a desired concentration, e.g., 100 ng/μl.

All kits described herein can further comprise devices to facilitate single-adminstration or repeated or frequent infusions of a synthetic, modified RNA, such as a non-implantable delivery device, e.g., needle, syringe, pen device, or an implantatable delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir. In some such embodiments, the delivery device can include a mechanism to dispense a unit dose of a composition comprising a synthetic, modified RNA. In some embodiments, the device releases the composition comprising a synthetic, modified RNA continuously, e.g., by diffusion. In some embodiments, the device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

Screening Methods

The ability to safely and efficiently reprogram, differentiate, transdifferentiate cells using the synthetic, modified RNAs compositions and methods thereof described herein, as well as generate engineered tissues using such cells, compositions and methods, has high applicability for use in high-throughput screening technologies of disease model systems and assays for the characterization of candidate agents for identifying novel agents for use in the treatment of human disease. Such screening methods and platforms can be used, for example, to identify novel agents for treating a desired disorder; to identify novel agents involved in reprogramming and differentiation, and/or alteration/maintenance of developmental states; or to identify effects of a candidate agent on one or more parameters of a particular cell type or engineered tissue generated using the compositions and methods described herein. Characterization of candidate agents can include aspects such as compound development, identifying cell-specific toxicity and cell-specific survival, and assessments of compound safety, compound efficacy, and dose-response parameters. For example, an engineered myocardium tissue can be contacted with a test agent, and the effect, if any, of the test agent on a parameter, such as an electrophysiological parameter, associated with normal or abnormal myocardium function, such as contractibility, including frequency and force of contraction, can be determined, or e.g., whether the agent has a cardiotoxic effect.

The drug discovery process is time-consuming and costly, in part owing to the high rate of attrition of compounds in clinical trials. Thus, modifications and alternative platforms that could accelerate the advancement of promising drug candidates, or reduce the likelihood of failure, would be extremely valuable. High-throughput screening technologies refer to the platforms and assays used to rapidly test thousands of compounds. For example, reporter systems used in cell lines can be used to assess whether compounds activate particular signaling pathways of interest.

The compositions and methods using synthetic, modified RNAs for reprogramming, differentiating, and transdifferentiating cells, as well as generating engineered tissues, described herein provide a reliable source of cells that can be generated and expanded in an efficient manner to quantities necessary for drug screening and toxicology studies. Further, because the compositions and methods comprising synthetic, modified RNAs described herein minimize the cellular interferon responses, and do not result in permanent genome modifications, the effects of a candidate agent can be studied with minimal confounding factors. As has been described herein, cells can be differentiated to generate specific cell types (for example, neurons, blood cells, pancreatic islet cells, muscle cells, and cardiomyocytes), and induced pluripotent stem cells can be generated from patients with specific diseases, such as, for example, a patient with cystic fibrosis, as demonstrated herein.

One particular advantage of cells and engineered tissues generated using the compositions, methods, and kits comprising synthetic, modified RNAs described herein for use in screening platforms, is that from a single and potentially limitless starting source, most of the major cells within the human body that could be affected by a drug or other agent can be produced. Such cells provide a better predictive model of both drug efficacy and toxicity than rodent cell lines or immortalized human cell lines that are currently used in high-throughput screens. While such immortalized cell and animal models have contributed a wealth of information about the complexity of various disease processes, compounds that show a significant benefit in such models can fail to show effectiveness in clinical trials. For example, use of a transgenic mouse that overexpresses mutant superoxide dismutase (SOD), a gene found to be associated with amyotrophic lateral sclerosis, enabled the identification of several compounds that alter disease characteristics, including vitamin E and creatine. However, when these compounds were tested in humans, no clinical improvements were observed (A. D. Ebert and C. N. Svendsen, “Human stem cells and drug screening: opportunities and challenges.” 2010 Nature Reviews Drug Discovery 9, p. 1-6). Furthermore, toxic effects of compounds are often missed in cell and animal models due to specific interactions with human biological processes that cannot be recapitulated in these systems.

Accordingly, in some aspects, the compositions comprising synthetic, modified RNAs, and the methods described herein, can be used for evaluating the effects of novel candidate agents and compounds on specific human cell types that are relevant to drug toxicity effects. In some embodiments, cells can be induced to undergo differentiation to a particular cell type or tissue, using the synthetic, modified RNAs described herein, that the test drug or compound is discovered or known to affect, and then used for performing dose-response toxicity studies. In such embodiments, human stem cells, such as iPS cells, derived from patients can be exposed to appropriate differentiation factors using the compositions and methods comprising synthetic, modified RNAs described herein, and instructed to form the various cell types found in the human body, which could then be useful for assessing multiple cellular parameters and characteristics upon exposure to a candidate agent or compound. For example, the cells could be used to assess the effects of drug candidates on functional cardiomyocytes, or on cardiomyocytes having a specific genetic mutation, because drug development is often stalled by adverse cardiac effects. Thus, measurable disruption of electrophysiological properties by known and novel agents and compounds can be assessed in a clinically relevant, consistent, and renewable cell source. Also, for example, such cells can be used to identify metabolic biomarkers in neural tissues derived from human stem cells after toxin exposure. Such embodiments allow potentially toxic compounds to be eliminated at an early stage of the drug discovery process, allowing efforts to be directed to more promising candidates. As another example, islet cells generated using the methods and compositions comprising synthetic, modified RNAs described herein can be used to screen candidate agents (such as solvents, small molecule drugs, peptides, polynucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of islet precursor cells and their various progeny. For example, islet cell clusters or homogeneous β cell preparations can be tested for the effect of candidate agents, such as small molecule drugs, that have the potential to up- or down-regulate insulin synthesis or secretion. The cells are combined with the candidate agent, and then monitored for change in expression or secretion rate of insulin, using, for example, RT-PCR or immunoassay of the culture medium.

In other aspects, the compositions comprising synthetic, modified RNAs, and the methods thereof described herein, are used in differentiation screens, i.e., for identifying compounds that increase self-renewal or differentiation, promote maturation, or enhance cell survival of cells, such as stem cells, differentiated cells, or cancer cells.

In other aspects, the compositions comprising the synthetic, modified RNAs, and the methods thereof, described herein, can be used to screen for drugs that may correct an observed disease phenotype. In such aspects, cells can be expanded, differentiated into the desired cell type using synthetic, modified RNAs, and then used to screen for drugs that may correct the observed disease phenotype. A candidate agent or drug can be used to directly contact the surface of a reprogrammed, differentiated, transdifferentiated cell population, or engineered tissue by applying the candidate agent to a media surrounding the cell or engineered tissue. Alternatively, a candidate agent can be intracellular as a result of introduction of the candidate agent into a cell.

As used herein, “cellular parameters” refer to quantifiable components of cells or engineered tissues, particularly components that can be accurately measured, most desirably in a high-throughput system. A cellular parameter can be any measurable parameter related to a phenotype, function, or behavior of a cell or engineered tissue. Such cellular parameters include, changes in characteristics and markers of a cell or cell population, including but not limited to changes in viability, cell growth, expression of one or more or a combination of markers, such as cell surface determinants, such as receptors, proteins, including conformational or posttranslational modification thereof, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, e.g. mRNA, DNA, global gene expression patterns, etc. Such cellular parameters can be measured using any of a variety of assays known to one of skill in the art. For example, viability and cell growth can be measured by assays such as Trypan blue exclusion, CFSE dilution, and ³H incorporation. Expression of protein or polyeptide markers can be measured, for example, using flow cytometric assays, Western blot techniques, or microscopy methods. Gene expression profiles can be assayed, for example, using microarray methodologies and quantitative or semi-quantitiative real-time PCR assays. A cellular parameter can also refer to a functional parameter, such as a metabolic parameter (e.g., expression or secretion of a hormone, such as insulin or glucagon, or an enzyme, such as carboxypeptidase), an electrophysiological parameter (e.g., contractibility, such as frequency and force of mechanical contraction of a muscle cell; action potentials; conduction, such as conduction velocity), or an immunomodulatory parameter (e.g., expression or secretion of a cytokine or chemokine, such as an interferon, or an interleukin; expression or secretion of an antibody; expression or secretion of a cytotoxin, such as perforin, a granzyme, and granulysin; and phagocytosis).

The “candidate agent” used in the screening methods described herein can be selected from a group of a chemical, small molecule, chemical entity, nucleic acid sequences, an action; nucleic acid analogues or protein or polypeptide or analogue of fragment thereof. In some embodiments, the nucleic acid is DNA or RNA, and nucleic acid analogues, for example can be PNA, pcPNA and LNA. A nucleic acid may be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide agent or fragment thereof, can be any protein of interest, for example, but not limited to; mutated proteins; therapeutic proteins; truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins of interest can be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. A candidate agent also includes any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments, the candidate agent is a small molecule having a chemical moiety. Such chemical moieties can include, for example, unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups, including macrolides, leptomycins and related natural products or analogues thereof. Candidate agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

Also included as candidate agents are pharmacologically active drugs, genetically active molecules, etc. Such candidate agents of interest include, for example, chemotherapeutic agents, hormones or hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof. Exemplary of pharmaceutical agents suitable for use with the screening methods described herein are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all of which are incorporated herein by reference in their entireties. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992), the contents of which is herein incorporated in its entirety by reference.

Candidate agents, such as chemical compounds, can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the candidate compounds for use in the screening methods described herein are known in the art and include, for example, those such as described in R. Larock (1989) Comprehensive Organic Transformations, VCH Publishers; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, the contents of each of which are herein incoporated in their entireties by reference.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233, the contents of each of which are herein incoporated in their entireties by reference.

Libraries of candidate agents can be presented in solution (e.g., Houghten (1992), Biotechniques 13:412-421), or on beads (Lam (1991), Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.), the contents of each of which are herein incoporated in their entireties by reference.

Polypeptides to be Expressed

Essentially any polypeptide can be expressed using the synthetic, modified, RNAs described herein. Polypeptides useful with the methods described herein include, but are not limited to, transcription factors, targeting moieties and other cell-surface polypeptides, cell-type specific polypeptides, differentiation factors, death receptors, death receptor ligands, reprogramming factors, and/or de-differentiation factors.

Transcription Factors

In some embodiments, a synthetic, modified RNA or composition thereof encodes for a transcription factor. As used herein the term “transcription factor” refers to a protein that binds to specific DNA sequences and thereby controls the transfer (or transcription) of genetic information from DNA to mRNA. In one embodiment, the transcription factor encoded by the synthetic, modified RNA is a human transcription factor, such as those described in e.g., Messina D M, et al. (2004) Genome Res. 14(10B):2041-2047, which is herein incorporated by reference in its entirety.

Some non-limiting examples of human transcription factors (and their mRNA IDs and sequence identifiers) for use in the aspects and embodiments described herein include those listed herein in Table 1 (SEQ ID NOs: 1-1428 and 1483-1501).

TABLE 1 Exemplary Human Transcription Factors SEQ Gene ID Abbrev ScriptSureID mRNA ID NO: Class Description AA125825 AA125825 1 Other AA634818 AA634818 2 Other AATF NM_012138 3 bZIP apoptosis antagonizing transcription factor AB002296 NT_033233:4 AB002296 4 Bromodomain AB058701 NT_025741:494 AB058701 5 ZnF-Other AB075831 NT_011139:311 AB075831 6 ZnF-C2H2 ABT1 NM_013375 7 Other activator of basal transcription 1 ADNP NM_015339 8 Homeobox activity-dependent neuroprotector AEBP2 NT_035211:21 NM_153207 9 ZnF-C2H2 AE(adipocyte enhancer)- binding protein 2 AF020591 NM_014480 10 ZnF-C2H2 zinc finger protein AF0936808 NM_013242 11 Other similar to mouse Gir3 or D. melanogaster transcription factor IIB AF5Q31 NM_014423 12 Structural ALL 1 fused gene from 5q31 AHR NM_001621 13 bHLH aryl hydrocarbon receptor AHRR NT_034766:39 NM_020731 14 Co-repressor aryl hydrocarbon receptor repressor AI022870 AI022870 15 Other catalytic subunit of DNA polymerase zeta AI352508 AI352508 16 Other Highly similar to DPOZ_HUMAN DNA POLYMERASE ZETA SUBUNIT AI569906 AI569906 17 ZnF-C2H2 Weakly similar to ZN42_HUMAN ZINC FINGER PROTEIN 42 AIRE NM_000383 18 ZnF-PHD autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) AK024238 NT_023124:29 AK024238 19 Homeobox AK056369 NT_034877:1 AK056369 20 ZnF-C2H2 AK057375 NT_008389:5 AK057375 21 ZnF-C2H2 AK074366 NT_005825:35 AK074366 22 ZnF-C2H2 AK074859 NT_011150:41 AK074859 23 ZnF-C2H2 AK092811 NT_017568:327 AK092811 24 ZnF-C2H2 AK096221 NT_035560:44 AK096221 25 ZnF-C2H2 AK096288 NT_007819:700 AK096288 26 ZnF-C2H2 AK098183 NT_011104:165 AK098183 27 ZnF-C2H2 AK122874 NT_011568:219 AK122874 28 ZnF-C2H2 AK126753 NT_011176:418 AK126753 29 ZnF-C2H2 ANP32A NM_006305 30 Co-activator phosphoprotein 32 family, member A APA1 NM_021188 31 ZnF-C2H2 ortholog of mouse another partner for ARF 1 Apg4B NM_013325 32 Other Apg4/Au2 homolog 2 (yeast) AR NM_000044 33 NHR androgen receptor (dihydrotestosterone receptor) ARC NM_015193 34 Other activity-regulated cytoskeleton-associated protein ARIDIA NT_028053:228 NM_006015 35 Structural AT rich interactive domain 1A (SWI-like) ARIH2 NM_006321 36 ZnF-Other ariedne (drosophila) homolog 2 ARIX NM_005169 37 Homeobox aristaless homeobox ARNT NM_001668 38 bHLH aryl hydrocarbon receptor nuclear translocator ARNT2 NM_014862 39 bHLH aryl hydrocarbon receptor nuclear translocator 2 ARNTL NM_001178 40 bHLH aryl hydrocarbon receptor nuclear translocator-like ARNTL2 NT_035213:171 NM_020183 41 bHLH aryl hydrocarbon receptor nuclear translocator-like 2 ARX NT_025940:10 NM_139058 42 Homeobox aristaless related homeobox ASCL1 NM_004316 43 bHLH achaete-scute complex (Drosophila) homolog-like 1 ASCL2 NM_005170 44 bHLH achaete-scute complex (Drosophila) homolog-like 2 ASCL3 NM_020646 45 bHLH achaete-scute complex (Drosophila) homolog-like 3 ASH1 NM_018489.2 46 ZnF-PHD hypothetical protein ASH1 ASH2L NM_004674 47 Structural Ash2 (absent, small, or homeotic, Drosophila, homolog)-like ATBF1 NM_006885 48 ZnF-C2H2 AT-binding transcription factor 1 ATF1 NM_005171 49 bZIP activating transcription factor 1 ATF2 NM_001880 50 bZIP activating transcription factor 2 ATF3 NM_001674 51 bZIP activating transcription factor 3 ATF4 NM_001675 52 bZIP Activating transcription factor 4 (tax-responsive enhancer element B67) ATF5 NM_012068 53 bZIP activating transcripton factor 5 ATF6 NM_007348 54 bZip activating transcription factor 6 AW875035 AW875035 55 AnF-C2H2 Moderately similar to YY1, Very very hypothetical protein RMSA-1 AWP1 NM_019006 56 ZnF-AN1 protein associated with PRK1 AY026053 NT_011519:29 AY026053 57 Heat Shock BA044953 NT_005825:31 AB079778.1 1497 OSZF isoform; ras- U26914.1 1498 responsive element binding protein (RREB-1) BACH1 NM_001186 58 bZIP BTB and CNC homology 1, basic leucine zipper transcription factor 1 BACH2 NM_021813 59 bZIP BTB and CNC homology 1, basic leucine zipper transcription factor 2 BAGE2 NT_029490:10 NM_182482 60 ZnF-PHD B melanoma antigen family, member 2 BANP NM_017869 61 Co-activator BANP homolog, SMAR1 homolog BAPX1 NM_001189 62 Homeobox bagpipe homeobox (Drosophila) homolog 1 BARHL1 NM_020064 63 Homeobox BarH (Drosophila)-like 1 BARHL2 AJ251753 64 Homeobox BarH (Drosophila)-like 2 BARX1 NM_021570 65 Homeobox BarH-like homeobox 1 BARX2 NM_003658 66 Homeobox BarH-like homeobox 2 BATF NM_006399.3 67 bZIP basic leucine zipper transcription factor, ATF- like BAZ1A NM_013448 68 Bromodomain bromodomain adjacent to zinc finger domain, 1A BAZ1B NM_023005 69 Bromodomain bromodomain adjacent to zinc finger domain, 1B BAZ2A NM_013449 70 Bromodomain bromodomain adjacent to zinc finger domain, 2A BAZ2B NM_013450.2 71 Bromodomain bromodomain adjacent to zinc finger domain, 2B BCL11A NM_018014 72 ZnF-C2H2 B-cell CLL/lymphoma 11A (zinc finger protein) BCL11B NM_022898 73 ZnF-C2H2 B-cell CLL/lymphoma 11B (zinc finger protein) BHLHB3 NM_030762 74 bHLH basic helix-loop-helix domain containing, class B, 3 BHLHB5 NM_152414 75 bHLH basic helix-loop-helix domain containing, class B, 5 BIA2 NT_029870:6 NM_015431 76 Co-activator BIA2 protein BIZF1 NM_003666 77 bZIP Basic leucine zipper nuclear factor 1 (JEM-1) BMI1 NM_005180 78 ZnF-Other murine leukemia viral (bmii-1) oncogene homolog BNC NM_001717 79 Znf-C2H2 basonuclin BRD1 NM_014577 80 Bromodomain bromodomain-containing 1 BRD2 NM_005104 81 Bromodomain bromodomain-containing 2 BRD3 NM_007371 82 Bromodomain bromodomain-containing 3 BRD4 NM_014299 83 Bromodomain bromodomain-containing 4 BRD7 NM_013263 84 Bromodomain bromodomain-containing 7 BRD9 NT_034766:148 NM_023924 85 Bromodomain bromodomain-containing 9 BRDT NM_001726 86 Bromodomain Bromodomain, testis- specific BRF1 NM_001519 87 Other BRF1 homolog, subunit of RNA polymerase III transcription initiation factor IIIB BRF2 NM_006887 88 ZnF-C3H zinc finger protein 36, C3H type-like 2 BRPF1 NM_004634 89 Bromodomain bromodomain and PHD finger containing, 1 BRPF3 AB033112 90 Bromodomain bromodomain and PHD finger containing, 3 BS69 NM_006624 91 ZnF-NYND Adenovirus 5 E1A binding protein BTAF1 AF038362 92 Other BTAF1 RNA polymerase II, B-TF11D transcription factor-associated, 170 kDa BTBD1 NT_019601:32 NM_025238 93 ZnF- BTB (POZ) domain BTB/POZ containing 1 BTBD14A NT_019501:127 NM_144653 94 ZnF- BTB (POZ) domain BTB/POZ containing 14A BTBD14B NT_031915:27 NM_052876 95 ZnF- BTB (POZ) domain BTB/POZ containing 14B BTBD2 NT_011268:135 NM_017797 96 ZnF- BTB (POZ) domain BTB/POZ containing 2 BTBD3 NM_014962 97 ZnF- BTB (POZ) domain BTB/POZ containing 3 BTBD4 NT_033241:138 AK023564 98 ZnF- BTB (POZ) domain BTB/POZ containing 4 BTF3L2 M90355 99 Other basic transcription factor 3, like 2 BTF3L3 N90356 100 Other Basic transcription factor 3, like 3 BX538183 NT_011109:1331 BX538183 101 ZnF-C2H2 BX548737 NT_006802:14 BX648737 102 ZnF-C2H2 C11orf13 NM_003475 103 Other chromosome 11 open reading frame 13 C11orf9 NM_013279 104 Other chromosome 11 open reading frame 9 C14orf101 NM_017799 105 Other chromosome 14 open reading frame 101 C14orf106 NM_018353 106 Other chromosome 14 open reading frame 106 C14orf44 NT_010422:242 NM_024731 107 ZnF- chromosome 16 open BTB/POZ reading frame 44 C1orf2 NM_006589 108 Other chromosome 10 open reading frame 2 C20orf174 AL713683 109 ZnF-C2H2 chromosome 20 open reading frame174 C21orf18 NM_017438 110 Other chromosome 21 open reading frame 18 C31P1 NT_034563:155 NM_021633 111 ZnF- kelch-like protein C31P1 BTB/POZ C5orf7 NM_016604 112 Jumonji chromosome 5 open reading frame 7 CART1 NM_006982 113 Homeobox cartilage paired-class homeoprotein 1 CBF2 NM_005760 114 Beta-scaffold- CCAAT-box-binding CCAAT transcription factor CBFA2T1 NM_004349 115 ZnF-MYND core-binding factor, runt domain, alpha subunit 2; translocated to, 1; cyclin D-related CBFA2T2 NT_028392:284 NM_005093 116 ZnF-MYND core-binding factor, runt domain, alpha subunit 2; translocated to, 2 CBFA2T3 NM_005187 117 ZnF-MYNC Core-binding factor, runt domain, alpha subunit 2; translocated to, 3 CBX1 NM_006807 118 Structural chromobox homolog 1 (Drosophila HP1 beta) CBX2 X77824 119 Structural chromobox homolog 2 (Drosophila Pc class)) CBX3 NM_007276 120 Structural chromobox homolog 3 (Drosophila HP1 gamma) CBX4 NM_003655 121 Structural chromobox homolog 4 (Drosophila Pc class) CBX5 NM_012117 122 Structural chromobox homolog 5 (Drosophila HP1 alpha) CBX6 NM_014292 123 Structural chromobox homolog 6 CBX7 NM_175709 124 Structural chromobox homolog 7a) CDX1 NM_001804 125 Homeobox caudal-type homeobox transcription factor 1 CDX2 NM_001265 126 Homeobox caudal-type homeobox transcription factor 2 CDX4 NM_005193 127 Homeobox caudal-type homeobox transcription factor 4 CEBPA NM_004364 128 bZIP CCAAT/enhancer binding protein (C/EBP), alpha CEBPB NM_005194 129 bZIP CCAAT/enhancer binding protein (C/EBP), beta CEBPD NM_005195 130 bZIP CCAAT/enhancer binding protein (C/EBP), delta CEBPE NM_001805 131 bZIP CCAAT/enhancer binding protein (C/EBP), epsilon CEBPG NM_001806 132 bZIP CCAAT/enhancer binding protein (C/EBP), gamma CECR6 Nm_031890 133 Bromodomain cat eye syndrome chromosome region, candidate 6 CERD4 NM_012074 134 ZnF-PHD D4, zinc and double PHD fingers, family 3 CEZANNE NM_020205 135 Co-repressor cellular zinc finger anti- NF-KappaB Cezanne CG9879 A1217897 Other CG9879 (fly) homolog CGI-149 NM_016079 137 Other CGI-149 protein CGI-85 NM_017635 138 Structural CGI-85 protein CGI-99 NM_016039 139 Other CGI-99 protein CHD1 NM_001270 140 Structural chromodomain helicase DNA binding protein 1 CHD1L NM_024568 141 Structural chromodomain helicase DNA binding protein 1- like CHD2 NM_001271 142 Structural chromodomain helicase DNA binding protein 2 CHD3 NM_001272 143 Structural chromodomain helicase DNA binding protein 3 CHD4 NM_001273 144 Structural chromodomain helicase DNA binding protein 4 CHD5 NM_015557 145 Structural chromodomain helicase DNA binding protein 5 CHD6 NM_032221 146 Structural chromodomain helicase DNA binding protein6 CHES1 NM_005197 147 Forkhead checkpoint suppressor 1 CHX10 XM_063425 148 Homeobox ceh-10 homeo domain containing homolog (C. elegans) CIZ1 NT_029366:585 NM_012127 149 ZnF-C2H2 Cip1-interacting zinc finger protein CLOCK NM_004898 150 bHLH Clock (mouse) homolog CNOT3 NM_014516 151 Other CCRA-NOT transcription complex, subunit 3 CNOT4 NM_013316 152 Other CCRA-NOT transcription complex, subunit 4 CNOT8 NM_004779 153 Other CCRA-NOT transcription complex, subunit 8 COPEB NM_001300 154 ZnF-C2H2 core promoter element binding protein COPS5 NM_006837 155 Co-activator COP9 constitutive photommphogenic homolog subunit 5 (Arabidopsis) CORO1A NM_007074 156 bZIP coronin, actin-binding protein, 1A CREB1 NM_004379 157 bZIP cAMP responsive element binding protein 1 CREB3 NM_006468 158 bZIP cAMP responsive element binding protein 3 (Inman) CREB3L1 NM_052854 159 bZIP cAMP responsive element binding protein 3-like 1 CREB3L2 NT_007933:5606 NM_194071 160 bZIP cAMP responsive element binding protein 3-like 2 CREB3L3 NT_011255:184 NM_032607 161 bZIP cAMP responsive element binding protein 3-like 3 CREB3L4 NT_004858:17 NM_130898 162 bZIP cAMP responsive element binding protein 3-like 4 CREB5 NM_004904 163 bZIP cAMP responsive element binding protein 5 CREBBP NM_004380 164 ZnPHD CREP binding protein (Rubinstein-Taybi syndrome) CREBL1 NM_004381 165 bZIP cAMP responsive element binding protein-like 1 CREBL2 NM_001310 166 bZIP cAMP responsive element binding protein-like 2 CREG NM_003851 167 Other Cellular repressor of EIA- stimulated genes CREM NM_001881 168 bZIP cAMP responsive element modulator CRIP1 NM_001311 169 Co-activator cysteine-rich protein 1 (intestinal) CRIP2 NM_001312 170 Co-activator cysteine-rich protein 2 CROC4 NM_006365 171 Other transcriptional activator of the c-fos promoter CRSP8 NM_004269 172 Co-activator cofactor required for Sp1 transcriptional activation, subunit 8, 34 kD CRSP9 NM_004270 173 Co-activator cofactor required for Sp1 transcriptional activation, subunit 9, 33 kD CRX NM_000554 174 Homeobox cone-rod homeobox CSDA NM_003651 175 Beta-scaffold- cold shock domain protein cold-shock A CSEN NM_013434 176 Other Calsenilin, presenilin- binding protein, EF hand transcription factor CSRP1 NM_004078 177 Co-activator cysteine and glycine-rich protein 1 CSRP2 NM_001321 178 Co-activator cysteine and glycine-rich protein 2 CSRP3 NM_003476 179 Co-activator cysteine and glycine-rich protein 3 (cardiac LIM protein) CTCF NM_006565 180 ZnF-C2H2 CCCTC-binding factor (zinc finger protein) CTCFL NT_011362:1953 NM_080618 181 ZnF-C2H2 CCCTC-binding factor (zinc finger protein)-like CTNNB1 NM_001904 182 Co-activator catenin (cadherin- associated protein), beta 1, 88 kD CUTL1 NM_001913 183 Homeobox cut (Drosophila)-like 1 (CCAAT displacement protein) CUTL2 AB006631 184 Homeobox cut-like 2 (Drosophila)- MAMLD1 NM_001177465.1 185 Other isoform 1 NM_001177466.1 1483 isoform 2 NM_005491.3 1484 isoform 3 DACH NM_004392 186 Co-repressor dachshund (Drosophila) homolog DAT1 NM_018640 187 ZnF-Other neuronal specific transcription factor DAT1 DATF1 NM_022105 188 ZnF-PHD death associated transcription factor 1 DBP NM_001352 189 bZIP D site of albumin promoter (albumin D-box) binding protein DDIT3 NM_004083 190 bZIP DNA-damage-inducible transcript 3 DEAF1 NM_021008 191 ZnF-MYND deformed epidermal autoregulatory factor 1 (Drosophila) DKFZP434 AB037779 192 Other DKFZP434B0335 protein B0335 DKFZP434 NM_031284 193 Other Hypothetical protein B195 DKFZp434B195 DKFZp434 AL080134 194 bHLH HLHmdelta (fly) homolog G043 DKFZP434 NM_015527 195 Other DKFZP434P1750 P1750 DKFZp547 NT_011233:43 NM_152606 196 ZnF-C2H2 Hypothetical protein B0714 DKFZp547B0714 DLX2 NM_004405 197 Homeobox Distal-less homeobox 2 DLX3 NM_005220 198 Homeobox distal-less homeobox 3 DLX4 NM_001934 199 Homeobox distal-less homeobox 4 DLX5 NM_005221 200 Homeobox distal-less homeobox 5 DLX6 NM_005222 201 Homeobox distal-less homeobox 6 DMRT1 NM_021951 202 ZnF-DM doublesex and mab-3 related transcription factor 1 DMRT2 NM_006557 203 ZnF-DM doublesex and mab-3 related transcription factor 2 DMRT3 NT_008413:158 NM_021240 204 ZnF-DM doublesex and mab-3 related transcription factor 3 DMRTA1 NT_023974:296 AJ290954 205 ZnF-DM DMRT-like family Al DMRTA2 AJ301580 206 ZnF-DM DMRT-like family A2 DMRTB1 NT_004424:223 NM_033067 207 ZnF-DM DMRT-like family B with protien-rich C-terminal, 1 DMRTC1 BC029799 208 ZnF-DM DMRT-like family C1 DMRTC2 NT_011139:240 NM_033052 209 ZnF-DM DMRT-like family C2 DMTF1 NM_021145 210 Other cyclin D binding Nyb-like transcription factor 1 DR1 NM_001938 211 Co-repressor down-regulator of transcription 1, TBP- binding (negative collector 2) DRAP1 NM_006442 212 Co repressor DR1-associated protein 1 (negative cofactor 2 alpha) DRIL1 NM_005224 213 Structural dead ringer (Drosophila)- like 1 DRIL2 NM_006465 214 Structural dead ringer (Drosophila)- like 2 (bright and dead ringer) DRPLA NM_001940 215 Co-repressor dentatorubral- palidoluysian atrophy (atrophin-1) DSIPI NM_004089 216 bZIP delta sleep inducing peptide, immunoreactor DTX2 AB040961 217 ZnF-other deltex homolog 2 (Drosophila) DUX1 NM_012146 218 Homeobox double homeobox 1 DUX2 NM_012147 219 Homeobox double homeobox 2 DUX3 NM_012148 220 Homeobox double homeobox genes 3 DUX4 NM_033178 221 Homeobox double homeobox 4 DUX5 NM_012149 222 Homeobox double homeobox 5 DXYS155E NM_005088 223 Other DNA segment on chromosome X and Y (unique) 155 expressed sequence E2F1 NM_005225 224 E2F E2F transcription factor 1 Text cut off EED NM_003797 225 Structural Embryonic echoderm development EGLN1 NT_004753:53 NM_022051 226 ZnF-MYND egl nine homolog 1 (C. elegans) EGLN2 NM_017555 227 ZnF-MYND egl nine homolog 2 (C. elegans) EGR1 NM_001964 228 ZnF-C2H2 early growth response 1 EGR2 NM_000399 229 ZnF-C2H2 early growth response 2 (Knox-20 (Drosophila) homolog) EGR3 NM_004430 230 ZnF-C2H2 early growth response 3 EGR4 NM_001965 231 ZnF-C2H2 early growth response 4 EHF NM_012153 232 Trp cluster- ets homologous factor Ets EHZF NT_011044:150 NM_015461 233 ZnF-PHD early hematopoietic zinc finger ELD/OSA1 NM_020732 234 Structural BRG1-binding protein ELD/OSA1 ELF1 M82882 235 Trp cluster E-74-like factor 1 (ets Ets domain transcription factor) ELF2 NM_006874 236 Trp cluster E-74-like factor 2 (ets Ets domain transcription factor) ELF3 NM_004433 237 Trp cluster E-74-like factor 3 (ets Ets domain transcription factor, epithelial-specific) ELF4 NM_001421 238 Trp cluster E-74-like factor 4 (ets Ets domain transcription factor) ELF5 NM_001422 239 Trp cluster E-74-like factor 5 (ets Ets domain transcription factor) ELK1 NM_005229 240 Trp cluster ELK1, member of ETS Ets oncogene family ELK3 NM_005230 241 Trp cluster ELK3, ETS-domain Ets protein (SRF accessory protein 2) ELK4 NM_021795 242 Trp cluster ELK4, ETS-domain Ets protein (SRF accessory protein 1) EME2 NT_010552:331 AK074080 243 ZnF- essential meiotic BTB/POZ endonuclease I homolog 2 (S. pombe) EMX1 X68879 244 Homeobox empty spiracles homolog 1 (Drosophila) EMX2 NM_004098 245 Homeobox empty spiracles homolog 2 (Drosophila) EN1 NM_001426 246 Homeobox engrailed homolog 1 EN2 NM_001427 247 Homeobox engrailed homolog 2 EC1 NT_oo6713:275 NM_003633 248 ZnF- ectodermal-neural cortex BTB/POZ (with BTB-like domain) ENO1 NM_001428 249 Other enolase 1 EOMES NM_005442 250 T-box Eomesodermin (Xenopus laevis) homolog ERCC3 NM_000122 251 Other excision repair cross- complementing rodent repair deficiency, complementation group 3 ERCC6 NM_000124 252 Other excision repair cross- complementing rodent repair deficiency, complementation group 6 ERF NM_006494 253 Trp cluster- Ets2 repressor factor Ets ERG NM_004449 254 Trp cluster- v-ets avian Ets erythroblastosis virus E26 oncogene related ESR1 NM_000125 255 NHR estrogen receptor 1 ESR2 NM_001437 256 NHR estrogen receptor 2 ESRRA NM_004451 257 NHR estrogen-related receptor alpha ESRRB NM_004452 258 NHR estrogen-related receptor beta ESRRG NM_001438 259 NHR estrogen-related receptor gamma ESXIL NT_01165135 NM_153448 260 Homeobox extraembryonic, spermatogenesis, homeobox 1-like ETR101 NM_004907 261 Other immediate early protein ETS1 NM_005238 262 Trp cluster- v-ets avian Ets erythroblastosis virus E26 oncogene homolog 1 ETS2 NM_005239 263 Trp cluster- v-ets avian Ets erythroblastosis virus E26 oncogene homolog 2 ETV1 NM_004956 264 Trp cluster- ets variant gene 1 Ets ETV2 AF000671 265 Trp cluster- ets variant gene 2 Ets ETV3 L16464 266 Trp cluster- ets variant gene3 Ets ETV4 NM_001986 267 Trp cluster- ets variant gene 4 (E1A Ets enhancer-binding protein, E1AF) ETV5 NM_004454 268 Trp cluster- ets variant gene 5 (ets- Ets related molecule) ETV6 NM_001987 269 Trp cluster- ets variant gene 6, TEL Ets oncogene EV11 NT_034563:55 NM_005241 270 ZnF-C2H2 ecotropic viral integration site 1 EVX1 NM_001989 271 Homeobox eve, even-skipped homeo box homolog 1 (Drosophila) EVX2 M59983 272 Homeobox eve, even-skipped homeo box homolog 2 (Drosophila) EYA1 NM_000503 273 Other eyes absent (Drosophila) homolog 1 EYA2 NM_005204 274 Other eyes absent (Drosophila) homolog 2 FBI1 NM_015898 275 ZnF- short transcripts binding BTB/POZ protein; lymphoma related factor FEM1A AL359589 276 Other fem-1 homolog a (C. elegans) FEZL NM_018008 277 ZnF-C2H2 likely ortholog of mouse and zebrafish forebrain embryonic zinc finger-like FHL1 NM_001449 278 ZnF-Other four and a half LIM domains 1 FHL2 NM_001450 279 ZnF-Other four and a half LIM domains 2 FHL5 NM_020482 280 Co-activator four and a half LIM domains 5 FHX NM_018416 281 Forkhead FOXJ2 forkhead factor FKHL18 AF042831 282 Forkhead forkhead (Drosophila)-like 18 FLI1 NM_002017 283 Trp cluster- friend leukemia virus Ets integration 1 FMR2 NM_002025 284 AF-4 fragile X mental retardation 2 FOS NM_005252 285 bZIP v-fos FBJ murine osteosarcoma viral oncogene homolog FOSB NM_006732 286 bZIP FBJ murine osteosarcoma viral oncogene homolog B FOSL1 NM_005438 287 bZIP FOS-like antigen 1 FOSL2 NM_005253 288 bZIP FOS-like antigen 2 FOXA1 NM_004496 289 Forkhead forkhead box A1 FOXA2 NM_021784 290 Forkhead forehead box A2 FOXE2 NM_012185 291 Forkhead forkhead box E2 FOXE3 NM_012186 292 Forkhead forkhead box E3 FOXF1 NM_001451 293 Forkhead forkhead box F1 FOXF2 NM_001452 294 Forkhead forkhead box F2 FOXG1B NM_005249 295 Forkhead forkhead box G1B FOXH1 NM_003923 296 Forkhead forkhead box H1 FOXI1 NM_012188 297 Forkhead forkhead box I1 FOXJ1 NM_001454 298 Forkhead forkhead box J1 FOXL1 NM_005250 299 Forkhead forkhead box L1 FOXL2 NM_023067 300 Forkhead forkhead box L2 FOXM1 NM_021953 301 Forkhead forkhead box M1 FOXN4 NT_009770:26 AF425596 302 Forkhead forkhead/winged helix transcription factor FOXN4 FOXO1A NM_002015 303 Forkhead forkhead box O1A (rhabdomyosarcoma) FOXO3A NM_001455 304 Forkhead forkhead box O3A FOXP1 AF275309 305 Forkhead forkhead box P1 FOXP2 NM_014491 306 Forkhead forkhead box P2 FOXP3 NM_014009 307 Forkhead forkhead box P3 FOXP4 NT_007592:3277 NM_138457 308 Forkhead forkhead box P4 FOXQ1 NM_033260 309 Forkhead forkhead box Q1 FREQ NT_029366:864 NM_014286 310 Other frequenin homolog (Drosophila) FUBP1 NM_003902 311 Other far upstream element- binding protein FUBP3 NT_008338:25 BC001325 312 Other far upstream element (FUSE) binding protein 3 GABPA NM_002040 313 Trp cluster- GA-binding protein Ets transcription factor, alpha subunit (60 kD) GABPB1 NM_005254 314 Co-activator GA-binding protein transcription factor, beta subunit 1 (53 kD) GABPB2 NM_016655 315 Trp cluster- GA-binding protein Ets transcription factor, beta subunit 2 (47 kD) GAS41 NM_006530 316 Structural glioma-amplified sequence-41 GASC1 AB018323 317 ZnF-PHD gene amplified in squamous cell carcinoma 1 GATA1 NM_002049 318 ZnF-GATA GATA-binding protein 1 (globin transcription factor 1) GATA2 NM_002050 319 ZnF-GATA GATA-binding protein 2 GATA3 NM_002051 320 ZnF-GATA GATA-binding protein 3 GATA4 NM_002052 321 ZnF-GATA GATA-binding protein 4 GATA5 NM_080473 322 ZnF-GATA GATA-binding protein 5 GATA6 NM_005257 323 ZnF-GATA GATA-binding protein 6 GBX1 L11239 324 Homeobox gastrulation brain homeobox 1 GBX2 NM_001485 325 Homeobox gastrulation brain homeobox 2 GFI1 NM_005263 326 ZnF-C2H2 growth factor independent 1 GFI1B NM_004188 327 ZnF-C2H2 growth factor independent 1B (potential regulator of CDKN1A, translocated in CML) GIOT-1 AB021641 328 ZnF-C2H2 gonadotropin inducible transcription repressor 1 GIOT-2 NM_016264 329 ZnF-C2H2 gonadotropin inducible transcription repressor-2 GL1 NM_005269 330 ZnF-C2H2 glioma-associated oncogene homolog (zinc finger protein) GLI2 NM_005270 331 ZnF-C2H2 GLI-Kruppel family member GLI2 GLI3 NM_000168 332 ZnF-C2H2 GLI-Kruppel family member GLI3 (Greig cephalopolysyndactyly syndrome) GLI4 NT_023684:15 NM_138465 333 ZnF-C2H2 GLI-Kruppel family member GLI4 GLIS2 NM_032575 334 ZnF-C2H2 Kruppel-like zinc finger protein GLIS2 GREB1 NT_005334:553 NM_014668 335 Co-repressor GREB1 protein GRLF1 NM_004491 336 ZnF-Other glucocorticoid receptor DNA binding factor 1 GSC NM_173849.2 337 Homeobox goosecoid GSCL NM_005315 338 Homeobox goosecoid-like GSH1 XM_046853 339 Homeobox genomic screened homeo box 1 homolog (mouse) GSH2 NM_133267 340 Homeobox genomic screened homeo box 2 homolog (mouse) GTF2A1 NM_015859 341 Other general transcription factor 11A, 1 (37 kD and 191 kD subunits) GTF2A2 NM_004492 342 Other general transcription factor IIA, 2 (12 kD subunit) GTF2B NM_001514 343 Other general transcription factor 11B GTF2E1 NM_005513 344 Other general transcription factor IIE, polypeptide 1 (alpha subunit, 56 kD) GTF2E2 NM_002095 345 Other general transcription factor IIE, polypeptide 2 (beta subunit, 34 kD) GTF2F1 NM_002096 346 Other general transcription factor IIF, polypeptide I (74 kD subunit) GTF2F2 NM_004128 347 Other general transcription factor IIF, polypeptide 2 (30 kD subunit) GTF2H1 NM_005316 348 Other general transcription factor IIH, polypeptide I (62 kD subunit) GTF2IRD1 NT_007758:1220 NM_005685 349 bHLH GTF21 repeat domain containing 1 GTF2IRD2 NT_007758:1320 NM_173537 350 bHLH transcription factor GTF2IRD2 GTF3A NM_002097 351 Other general transcription factor IIIA GTF3C1 NM_001520 352 Other general transcription factor IIIC, polypeptide 1 (alpha subunit, 220 kD) GTF3C2 NM_001521 353 Other general transcription factor IIIC, polypeptide 2 (beta subunit, 110 kD) GTF3C3 NM_012086 354 Other general transcription factor IIIC, polypeptide 3 (102 kD) GTF3C4 NM_012204 355 Other general transcription factor IIIC, polypeptide 4 (90 kD) GTF3C5 NM_012087 356 Other general transcription factor IIIC, polypeptide 5 (63 kD) HAND1 NM_004821 357 bHLH heart and neural crest derivatives expressed 1 HAND2 NM_021973 358 bHLH basic helix-loop-helix transcription factor HAND2 HATH6 NT_015805:94 NM_032827 359 bHLH basic helix-loop-helix transcription factor 6 HBOA NM_007067 360 Co-activator histone acetyltransferase HCF2 NM_013320 361 Other host cell factor 2 HCNGP NM_013260 362 Other transcriptional regulator protein HDAC1 NM_004964 363 Co-repressor histone deacetylase 1 HDAC2 NM_001527 364 Co-repressor histone deacetylase 2 HDAC4 NM_006037 365 Co-repressor histone deacetylase 4 HDAC8 NT-011594:18 NM_018486 366 Structural histone deacetylase 8 HES2 NM_019089 367 bHLH hairy and enhancer of split 2 (Drosophila) HES5 BQ924744 368 bHLH hairy and enhancer of split 5 (Drosophila) HES6 NM_018645 369 bHLH hairy and enhancer of split 6 (Drosophila) HES7 NM_032580 370 bHLH hairy and enhancer of split 7 (Drosophila) HESX1 NM_003865 371 Homeobox homeobox (expressed in ES cells) 1 HEY1 NM_012258 372 bHLH hairy/enhancer-of-split related with YRPW motif 1 (‘YRPW’ disclosed as SEQ ID NO: 1482) HEY2 NM_012259 373 bHLH hairy/enhancer-of-split related with YRPW motif 2 (‘YRPW’ disclosed as SEQ ID NO: 1482) HEYL NM_014571 374 bHLH hairy/enhancer-of-split related with YRPW motif- life (‘YRPW’ disclosed as SEQ ID NO: 1482) HHEX NM_002729 375 Homeobox hematopoietically expressed homeobox cutoff HIVEP1 NM_002114 376 ZnF-C2H2 human immunodeficiency virus type I enhancer- binding protein 1 HIVEP2 NM_006734 377 ZnF-C2H2 human immunodeficiency virus type I enhancer- binding protein 2 HIVEP3 NT_004852:421 NM_024503 378 ZnF-C2H2 human immunodeficiency virus type 1 enhancer binding protein 3 HKR1 BC004513 379 ZnF-C2H2 GLI-Kruppel family member HKR1 HKR2 M20676 380 ZnF-C2H2 GL1-Kruppel family member HKR2 HKR3 NM_005341 381 ZnF- GLI-Kruppel family BTB/POZ member HKR3 HLF NM_002126 382 bZIP hepatic leukemia factor HLX1 NM_021958 383 Homeobox H2.0 (Drosophila)-like homeo box 1 HLXB9 NM_005515 384 Homeobox homeo box HB9 HMG20A NT_024654:319 NM_018200 385 Structural high-mobility group 20A HMG20B NM_006339 386 Structural high-mobility group 20B HMGA1 NM_002131 387 Beta-scaffold- high mobility group AT- HMG hook 1 HMGA2 NM_003483 388 Beta-scaffold- high mobility group AT- HMG hook 2 HMGB1 NM_002128 389 Structural high-mobility group box 1 HMGB2 NM_002129 390 Structural high-mobility group box 2 HMGB3 NT_011602:55 NM_005342 391 Structural high-mobility group box 3 HMGN2 NM_005517 392 Structural high-mobility group nucleosomal binding domain 2 HMX1 NM_018942 393 Homeobox homeo box (H6 family) 1 HMX2 NM_005519.1 394 Homeobox homeo box (H6 family) 2 HMX3 XM_114950 395 Homeobox homeo box (H6 family) 3 HNF4A NM_000457 396 NHR hepatocyte nuclear factor 4, alpha HNF4G NM_004133 397 NHR hepatocyte nuclear factor 4, gamma HOP NM_032495 398 Homeobox homeodomain-only protein HOXA1 NM_005522 399 Homeobox homeobox A1 HOXA10 NM_018951 400 Homeobox homeobox A10 HOXA11 NM_005523 401 Homeobox homeobox A11 HOXA13 NM_000522 402 Homeobox homeobox A13 HOXA2 NM_006735 403 Homeobox homeobox A2 HOXA3 NM_030661 404 Homeobox homeobox A3 HOXA4 NM_002141 405 Homeobox homeobox A4 HOXA5 NM_019102 406 Homeobox homeobox A5 HOXB9 NM_024017 407 Homeobox homeobox B9 HOXC10 NM_017409 408 Homeobox homeobox C10 HOXC11 NM_014212 409 Homeobox homeobox C11 HOXC12 X99631 410 Homeobox homeobox C12 HOXC13 NM_017410 411 Homeobox homeobox C13 HOXC4 NM_014620 412 Homeobox homeobox C4 HOXC5 NM_018953 413 Homeobox homeobox C5 HOXC6 NM_004503 414 Homeobox homeobox C6 HOXC8 NM_022658 415 Homeobox homeobox C8 HOXC9 NM_006897 416 Homeobox homeobox C9 HOXD1 NM_024501 417 Homeobox homeobox D1 HOXD10 NM_002148 418 Homeobox homeobox D10 HOXD11 NM_021192 419 Homeobox homeobox D11 HOXD12 NM_021193 420 Homeobox homeobox D12 HOXD13 NM_000523 421 Homeobox homeobox D13 HOXD3 NM_006898 422 Homeobox homeobox D3 HOXD4 NM_014621 423 Homeobox homeobox D4 HOXD8 NM_019558 424 Homeobox homeobox D8 HOXD9 NM_014213 425 Homeobox homeobox D9 HPCA NT_00451193 NM_002143 426 Other hippocalcin HPCAL1 NT_005334:412 NM_002149 427 Other hippocalcin-like 1 H-plk NM_015852 428 ZnF-C2H2 Krueppel-related zinc finger protein HR AF039196 429 Jumonji hairless HRIHFB2122 NM_007032 430 Other Tara-like protein (Drosophila) HRY NM_005524 431 bHLH hairy (Drosophila)-homolog HS747E2A NM_015370 432 Other hypothetical protein (RING domain) HSA275986 NM_018403 433 Other transcription factor SMIF HSAJ2425 NM_017532 434 NHR p65 protein HSF1 NM_005526 435 Heat shock Heat shock transcription factor 1 HSF2 NM_004506 436 Heat shock Heat shock transcription factor 2 HSF2BP NM_007031 437 Co-activator Heat shock transcription factor 2 binding protein HSF4 NM_001538 438 Heat shock Heat shock transcription factor 4 HSFY NM_033108 439 Heat shock Heat shock transcription factor, Y-linked HSGT1 NM_007265 440 Other suppressor of S. cerevisiae gcr2 HSHPX5 X74862 441 Other HPX-5 HSPC018 NM_014027 442 Other HSPC018 protein HSPC059 NT_011233:37 NM_016536 443 ZnF-C2H2 HSPC059 protein HSPC063 NT_033899:972 NM_014155 444 ZnF-C2H2 HSPC063 protein HSPC189 NM_016535 445 Other HSPC189 protein HSPX153 X76978 446 Homeobox HPX-153 homeobox HSRNAFEV NT_005403:123 NM_017521 447 Trp Cluster- FEV protein Ets HSU79252 NM_013298 448 Other hypothetical protein ID1 NM_002165 449 bHLH inhibitor of DNA binding 1, negative helix-loop- helix protein ID2 NM_002166 450 bHLH inhibitor of DNA binding 2, dominant negative helix-loop-helix protein ID2B NT_005999:169 M96843 451 bHLH inhibitor of DNA binding 2B, dominant negative helix-loop-helix protein ID3 NM_002167 452 bHLH inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ID4 NM_001546 453 bHLH inhibitor of DNA binding 4, dominant negative helix-loop-helix protein IGHMBP2 NM_002180 454 ZnF-AN1 immunoglobulin mu binding protein 2 ILF1 NM_004514 455 Forkhead interleukin in enhancer binding factor 1 ILF2 NM_004515 456 ZnF-C2H2 interleukin enhancer binding factor 2, 45 kDa ILF3 NM_012218 457 ZnF-C2H2 interleukin enhancer binding factor, 3, 90 kDa INSM1 NM_002196 458 ZnF-C2H2 insulinoma-associated 1 INSM2 NM_032594 459 ZnF-C2H2 insulinoma-associated protein 1A-6 IPF1 NM_000209 460 Homeobox insulin promoter factor 1, homeodomain transcription factor IRF1 NM_002198 461 Trp cluster- interferon regulatory IRF factor 1 IRF2 NM_002199 462 Trp cluster- interferon regulatory IRF factor 2 IRF3 NM_001571 463 Trp cluster- interferon regulatory IRF factor 3 IRF4 NM_002460 464 Trp cluster- interferon regulatory IRF factor 4 IRF5 NM_002200 465 Trp cluster- interferon regulatory IRF factor 5 IRF6 NM_006147 466 Trp cluster- interferon regulatory IRF factor 6 IRF7 NM_001572 467 Trp cluster- interferon regulatory IRF factor 7 IRLB X63417 468 Other c-myc promoter-binding protein IRX1 U90307 469 Homeobox iroquois homeobox protein 1 IRX2 AF319967 470 Homeobox iroquois homeobox protein 2 IRX3 U90308 471 Homeobox iroquois homeobox protein 3 IRX4 NM_016358 472 Homeobox Iroquois homeobox protein 4 IRX5 NM_005853 473 Homeobox Iroquois homeobox protein 5 IRX6 U90305 474 Homeobox Iroquois homeobox protein 6 JARID1A NT_009759:29 NM_005056 475 Jumonji Jumonji, AT rich interactive domain 1A (RBP2-like) JARID1B NT_034408:191 NM_006618 476 Jumonji Jumonji, AT rich interactive domain 1B (RBP2-like) JARID1D NT_011875:152 NM_004653 477 Jumonji Jumonji, AT rich interactive domain 1D (RBP2-like) JDP2 NT_026437:1173 NM_130469 478 bZIP jun dimerization protein 2 JMJ NM_004973 479 Jumonji jumonji homolog (mouse) JMJD1 NT_015805:184 NM_018433 480 Jumonji jumonji domain containing 1 JMJD2 NT_032971:21 BC002558 481 Jumonji jumonji domain containing 2 JMJD2B NT_011255:298 AK026040 482 Jumonji jumonji domain- containing 2B JUN NM_002228 483 bZIP v-jun avan sarcoma virus 17 oncogene homolog JUNB NM_002229 484 bZIP Jun B proto-oncogene JUND NM_005354 485 bZIP Jun D proto-oncogene KBTBD10 NT_005332:189 NM_006063 486 ZnF- kelch repeat and BTB BTB/POZ (POZ) domain containing 10 KBTBD5 NT_005825:210 NM_152393 487 ZnF- kelch repeat and BTB BTB/POZ (POZ) domain containing 5 KBTBD7 NT_009984:758 NM_032138 488 ZnF- kelch repeat and BTB BTB/POZ (POZ) domain containing 7 KCNIP1 NT_023132:191 NM_014592 489 Other Kv channel interacting protein 1 KCNIP2 NT_030059:932 NM_014591 490 Other Kv channel interacting protein 2 KCNIP4 NT_006344:469 NM_025221 491 Other Kv channel interacting protein 4 KEAP1 NM_012289 492 Other Kelch-like ECH- associated protein 1 KLF1 NM_006563 493 ZnF-C2H2 Kruppel-like factor 1 (erythroid) KLF12 NM_007249 494 ZnF-C2H2 Kruppel-like factor 12 KLF13 NM_015995 495 ZnF-C2H2 Kruppel-like factor 13 KLF14 NM_138693 496 ZnF-C2H2 Kruppel-like factor 14 KLF15 NM_014079 497 ZnF-C2H2 Kruppel-like factor 15 KLF16 NM_031918 498 ZnF-C2H2 Kruppel-like factor 16 KLF2 NM_016270 499 ZnF-C2H2 Kruppel-like factor 2 (lung) KLF3 NM_016531 500 ZnF-C2H2 Kruppel-like factor 3 (basic) KLF4 NM_004235 501 ZnF-C2H2 Kruppel-like factor 4 (gut) KLF5 NM_001730 502 ZnF-C2H2 Kruppel-like factor 5 (intestinal) KLF7 NM_003709 503 ZnF-C2H2 Kruppel-like factor 7 (ubiquitous) KLF8 NM_007250 504 ZnF-C2H2 Kruppel-like factor 8 KLHL1 NT_024524:413 NM_020866 505 ZnF- kelch-like 1 (Drosophila) BTB/POZ KLHL3 NT_016714:116 NM_017415 506 ZnF- kelch-like 3 (Drosophila) BTB/POZ KLHL4 NT_011689:82 NM_019117 507 ZnF- kelch-like 4 (Drosophila) BTB/POZ KLHL5 NM_015990 508 ZnF- kelch-like 5 (Drosophila) BTB/POZ KLHL6 NT_022676:150 NM_130446 509 ZnF- kelch-like 6 (Drosophila) BTB/POZ KLHL8 NT_006204:183 NM_020803 510 ZnF- kelch-like 8 BTB/POZ LDB1 NM_003893 511 Co-activator LIM domain binding 1 LDB2 NM_001290 512 Co-activator LIM domain binding 2 LDOC1 NM_012317 513 bZIP leucine zipper, down- regulated in cancer 1 LEF1 NM_016269 514 Beta-scaffold- lymphoid enhancer factor HMG 1 LHX1 NM_005568 515 Homeobox LIM homeobox protein 1 LHX2 NM_004789 516 Homeobox LIM homeobox protein 2 LHX3 NM_014564 517 Homeobox LIM homeobox protein 3 LHX4 NM_033343 518 Homeobox LIM homeobox protein 4 LHX5 NM_022363 519 Homeobox LIM homeobox protein 5 LHX6 NM_014368 520 Homeobox LIM homeobox protein 6 LHX8 AB050476 521 Homeobox LIM homeobox protein 8 LHX9 AJ277915 522 Homeobox LIM homeobox protein 9 LIM NM_006457 523 Co-activator LIM protein (similar to rat protein kinase C-binding enigma) LIN28 NM_024674 524 Beta-scaffold- RNA-binding protein LIN- cold-shock 28 LISCH7 NM_015925 525 bHLH liver-specific bHLH-Zip transcription factor LMO1 NM_002315 526 ZnF-Other LIM domain only 1 (rhombotin 1) LMO2 NM_005574 527 ZnF-Other LIM domain only 2 (rhombotin-like 1) LMO4 NM_006769 528 ZnF-Other LIM domain only 4 LMO6 NM_006150 529 ZnF-Other LIM domain only 6 LMO7 NM_005358 530 ZnF-Other LIM domain only 7 LMX1A AY078398 531 Homeobox LIM homeobox transcription factor 1, alpha LMX1B NM_002316 532 Homeobox LIM homeobox transcription factor 1, beta LOC113655 BC011982 533 Other hypothetical protein BC011982 LOC115468 NT_035560:126a NM_145326 534 ZnF-C2H2 similar to hypothetical protein FLJ13659 LOC115509 NT_024802:36 NM_138447 535 ZnF-C2H2 hypothetical protein BC014000 LOC115950 NT_011176:403 NM_138783 536 ZnF-C2H2 hypothetical protein BC016816 LOC126295 NT_011255:1 NM_173480 537 ZnF-C2H2 hypothetical protein LOC126295 LOC146542 NT_024802:32a NM_145271 538 ZnF-C2H2 similar to hypothetical protein MGC13138 LOC148213 NT_033317:111 NM_138286 539 ZnF-C2H2 hypothetical protein FLJ31526 LOC151162 AF055029 540 Other hypothetical protein LOC151162 LOC283248 NT_033241:294 NM_173587 541 Trp Cluster- hypothetical protein Myb LOC283248 LOC284346 NT_011109:18 NM_174945 542 ZnF-C2H2 hypothetical protein LOC284346 LOC285346 NT_034534:55 BC014381 543 Methyl-CpG- hypothetical protein binding LOC285346 LOC286103 NT_031818:174 NM_178535 544 ZnF-C2H2 hypothetical protein LOC286103 LOC51036 NM_015854 545 Other retinoic acid receptor-beta associated open reading frame LOC51042 NM_015871 546 ZnF-C2H2 zinc finger protein LOC51045 NM_015877 547 ZnF-C2H2 Kruppel-associated box protein LOC51058 NM_015911 548 ZnF-C2H2 hypothetical protein LOC51123 NM_016096 549 ZnF-C2H2 HSPC038 protein LOC51186 NM_016303 550 Other pp21 homolog LOC51193 NM_016331 551 ZnF-C2H2 zinc finger protein ANC_2H01 LOC51270 NM_016521 552 E2F E2F-like protein LOC51290 NM_016570 553 Other CDA14 LOC51333 NT_024802:6 NM_016643 554 ZnF-C2H2 mesenchymal stem cell protein DSC43 LOC55893 NM_018660 555 ZnF-C2H2 papillomavirus regulatory factor PRF-1 LOC56270 NM_019613 556 Other hypothetical protein 628 LOC56930 AL365410 557 Other hypothetical protein from EUROIMAGE 1669387 LOC57209 AJ245587 558 ZnF-C2H2 Kruppel-type zinc finger protein LOC57801 NM_021170 559 bHLH hairy and enhancer of split 4 (Drosophila) LOC58500 X16282 560 ZnF-C2H2 zinc finger protein (clone 647) LOC65243 NM_023070 561 ZnF-C2H2 hypothetical protein LOC86614 NM_033108 562 Heat shock Heat shock transcription factor 2-like LOC90322 AK001357 563 ZnF-C2H2 similar to KRAB zinc finger protein KR18 LOC90462 AK027873 564 ZnF-C2H2 similar to Zinc finger protein 84 (Zinc finger protein HPF2) LOC90589 NT_011176:506 NM_145233 565 bZIP similar to Zinc finger protein 20 (Zinc finger protein KOX13) LOC90987 AK000435 566 ZnF-C2H2 similar to ZINC FINGER PROTEIN 184 LOC91120 NM_033196 567 ZnF-C2H2 similar to ZINC FINGER PROTEIN 85 (ZINC FINGER PROTEIN HPF4) (HTF1) (H. sapiens) LOC91464 NT_011520:1976 AK025181 568 Homeobox hypothetical protein LOC91464 LOC91614 AJ245600 569 Other novel 58.3 KDA protein M96 NM_007358 570 ZnF-PHD likely ortholog of mouse metal response element binding transcription factor 2 MAD NM_002357 571 bHLH MAX dimerization protein 1 MADH1 NM_005900 572 Dwarfin MAD, mothers against decapentaplegic homolog 1 (Drosophila) MADH2 NM_005901 573 Dwarfin MAD, mothers against decapentaplegic homolog 2 (Drosophila) MADH3 NM_005902 574 Dwarfin MAD, mothers against decapentaplegic homolog 3 (Drosophila) MADH4 NM_005359 575 Dwarfin MAD, mothers against decapentaplegic homolog 4 (Drosophila) MADH5 NM_005903 576 Dwarfin MAD, mothers against decapentaplegic homolog 5 (Drosophila) MADH6 NM_005585 577 Dwarfin MAD, mothers against decapentaplegic homolog 6 (Drosophila) MADH7 NM_005904 578 Dwarfin Mad, mothers against decapentaplegic homolog 7 (Drosophila) MADH9 NM-005905 579 Dwarfin MAD, mothers against decapentaplegic homolog 9 (Drosophila) MAF NM_005360 580 bZIP v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAFB NM_005461 581 bZIP v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian) MAFF NM_012323 582 bZIP v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) MAFG NM_002359 583 bZIP v-maf musculoaponeurotic fibrosarcoma oncogene family, protein G (avian) v-maf musculoaponeurotic MBD4 NM_003925 584 Methyl-CpG- methyl-CpG binding binding domain protein 4 MBNL2 NM_005757 585 ZnF-C3H muscleblind-like 2 (Drosophila) MDS032 NM_018467 586 Other uncharacterized hematopoietic stem/progenitor cells protein MDS032 MDS1 NM_004991 587 Other myelodysplasia syndrome 1 MECP2 NM_004992 588 Methyl-CpG- methyl CpG binding binding protein 2 (Rett syndrome) MED6 NM_005466 589 Co-activator mediator of RNA polymerase II transcription, subunit 6 homolog (yeast) MEF2A NM_005587 590 Beta-scaffold- MADS box transcription MADS enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) MEF2B NM_005919 591 Beta-scaffold- MADS box transcription MADS enhancer factor 2, polypeptide B (myocyte enhancer factor 2B) MEF2C NM_002397 592 Beta-scaffold- MADS box transcription MADS enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) MEF2D NM_005920 593 Beta-scaffold- MADS box transcription MADS enhancer factor 2, polypeptide D (myocyte enhancer factor 2D) MEFV NM_000243 594 Co-activator Mediterranean fever (pyrin) MEIS1 NM_002398 595 Homeobox Meis1, myeloid ecotropic viral integration site 1 homolog (mouse) MEIS2 NM_020149 596 Homeobox Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse) MEIS3 U68385 597 Homeobox Meis1, myeloid ecotropic viral integration site 1 homolog 3 (mouse) MEOX1 NM_004527 598 Homeobox mesenchyme homeobox 1 MEOX2 NM_005924 599 Homeobox mesenchyme homeobox 2 (growth arrest-specific homeo box) MESP1 NT_033276:146 NM_018670 600 bHLH mesoderm posterior 1 MESP2 AL360139 601 bHLH mesoderm posterior 2 METTL3 NM_019852 602 Other methyltransferase like 3 MGA AB011090 603 bHLH MAX gene associated MHC2TA NM_000246 604 Other MHC class II transactivator MID1 NM_000381 605 Structural midline 1 (Opitz/BBB syndrome) MID2 NT_011651:146 NM_012216 606 Structural midline 2 MI-ER1 NM_020948 607 Other mesoderm induction early response 1 MILD1 NM_031944 608 Homeobox Mixl homeobox-like 1 (Xenopus laevis) MITF NM_000248 609 bHLH microphthalmia-associated transcription factor MLLT1 NM_005934 610 AF-4 myeloid/lymphoid or mixed-lineage leukemia (thrithorax (Drosophila) homolog); translocated to, 1 MLLT10 NM_004641 611 ZnF-PHD myeloid/lymphoid or mixed-lineage keukemia (trithorax (Drosophila) homolog); translocated to 10 MLLT2 NM_005935 612 AF-4 myeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila) homolog); translocated to 2 MLLT3 NM_004529 613 AF-4 myleloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila) homolog); translocated to 3 MLLT4 NM_005936 614 Structural myeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila) homolog); translocated to 4 MLLT6 NM_005937 615 ZnF-PHD myeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila) homolog); translocated to 6 MLLT7 NM_005938 616 Forkhead myeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila) homolog); translocated to, 7 MNAT1 NM_002431 617 ZnF-Other menage a trois 1 (CAK assembly factor) MNDA NM_002432 618 Other myeloid cell nuclear differentiation antigen MNT NM_020310 619 bHLH MAX binding protein MONDOA NM_014938 620 bHLH Mix interactor MORF NM_012330 621 ZnF-PHD monocytic leukemia zinc finger protein-related facto MORF4 NM_006792 622 Structural mortality factor 4 MORF4L1 NM_006791 623 Structural mortality factor 4 like 1 MORF4L2 NM_012286 624 Structural mortality factor 4 like 2 MRF-1 BC032488 625 Structural modulator recognition factor 1 MRF2 BC015120 626 Structural modulator recognition factor 2 MRG2 AL359938 627 Homeobox likely ortholog of mouse myeloid ecotropic viral integration site-related gene 2 MTF1 NM_005955 628 ZnF-C2H2 [cut off] transcription factor 1 MXD3 NM_031300 629 bHLH MAX dimerization protein 3 MXD4 NM_006454 630 bHLH MAX dimerization protein 4 MXI1 NM_005962 631 bHLH MAX interacting protein 1 MYB NM_005375 632 Trp cluster- v-myb myeloblastosis Myb. viral oncogene homolog (avian) MYBBP1A NM_014520 633 Co-repressor MYB binding protein (P160) 1a MYBL1 X66087 634 Trp cluster- v-myb myeloblastosis Myb viral oncogene homolog (avian)-like 1 MYBL2 NM_002466 635 Trp cluster- v-myb myeloblastosis Myb viral oncogene homolog (avian)-like 2 MYC NM_002467 636 bHLH v-myc myelocytomatosis (c-MYC) viral oncogene homolog (avian) MYCBP NM_012333 637 Co-activator c-myc binding protein MYCL1 M19720 638 bHLH v-myc myelocytomatosis viral oncogene homolog, lung carcinoma derived (arivan) MYCL2 NM_005377 639 bHLH v-myc myelocytomatosis viral oncogene homolog 2 (avian) MYCLK1 M64786 640 bHLH v-myc myelocytomatosis viral oncogene homolog (avian)-like 1 MYCN NM_005378 641 bHLH v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) MYF5 NM_005593 642 bHLH myogenic factor 5 MYF6 NM_002469 643 bHLH myogenic factor 6 (herculin) MYNN NT_010840:25 NM_018657 644 ZnF- myoneurin BTB/POZ MYOD1 NM_002478 645 bHLH myogenic factor 3 MYOG NM_002479 646 bHLH myogenin (myogenic factor 4) MYT1 NM_004535 647 ZnF-Other myelin transcription factor 1 MYT1L AF036943 648 ZnF-Other myelin transcription factor 1-like MYT2 NM_003871 649 Other myelin transcription factor 2 NAB1 NM_005966 650 Co-repressor NGFI-A binding protein 1 (EGR1 binding protein 1) NAB2 NM_005967 651 Co-repressor NGFI-A binding protein 2 (EGR1 binding protein 2) NCALD NM_032041 652 Other neurocalcin delta NCOA1 NM_003743 653 Co-activator nuclear receptor NCYM NM_006316 654 Other transcriptional activator NEUD4 NM_004647 655 ZnF-PHD Neuro-d4 (rat) homolog NEUROD1 NM_002500 656 bHLH neurogenic differentiation 1 NEUROD2 NM_006160 657 bHLH neurogenic differentiation 2 NEUROD4 NM_021191 658 bHLH neurogenic differentiation 4 NEUROD6 NM_022728 659 bHLH neurogenic differentiation 6 NEUROG1 NM_006161 660 bHLH neurogenin 1 NEUROG2 AF303002 661 bHLH neurogenin 2 NEUROG3 NM_020999 662 bHLH neurogenin 3 NFAT5 NM_006599 663 Beta-scaffold- nuclear factor of activated RHD T-cells 5, tonicity- responsive NFATC1 NM_006162 664 Beta-scaffold- nuclear factor of activated RHD T-cells, cytoplasmic, calcineurin-dependent 1 NFATC2 NM_012340 665 Beta-scaffold- nuclear factor of activated RHD T-cells, cytoplasmic, calcineurin-dependent 2 NFATC3 NM_004555 666 Beta-scaffold- nuclear factor of activated RHD T-cells, cytoplasmic, calcineurin-dependent 3 NFATC4 NM_004554 667 Beta-scaffold- nuclear factor of activated RHD T-cells, cytoplasmic, calcineurin-dependent 4 NFE2 NM_006163 668 bZIP nuclear factor (erythroid- derived 2), 45 kD NFE2L1 NM_003204 669 bZIP nuclear factor (erythroid- derived 2)-like 1 NFE2L2 NM_006164 670 bZIP nuclear factor (erythroid- derived 2)-like 2 NFE2L3 NM_004289 671 bZIP nuclear factor (erythroid- derived 2)-like 3 NFIA AB037860 672 Beta-scaffold- nuclear factor I/A CCAAT NFIB NM_005596 673 Beta-scaffold- nuclear factor I/B CCAAT NFIC NM_005597 674 Beta-scaffold- nuclear factor I/C CCAAT (CCAAT-binding transcription factor) NFIL3 NM_005384 675 bZIP nuclear factor, interleukin 3 regulated NFIX NM_002501 676 Beta-scaffold- nuclear factor I/X CCAAT (CCAAT-binding transcription factor) NFKBIB NM_002503 677 Co-activator kappa light polypeptide gene enhancer in B-cells inhibitor, beta NFKBIE NM_004556 678 Co-repressor nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon NFKBIL1 NM_005007 679 Co-repressor nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 NFKBIL2 NM_013432 680 Co-repressor nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 2 NFRKB NM_006165 681 Beta-scaffold- nuclear factor related to RHD kappa B binding protein NFX1 NM_002504 682 RFX nuclear transcription factor, X-box binding 1 NFYA NM_002505 683 Beta-scaffold- nuclear transcription factor CCAAT Y, alpha NFYB NM_006166 684 Beta-scaffold- nuclear transcription factor CCAAT Y, beta NFYC NM_014223 685 Beta-scaffold- nuclear transcription factor CCAAT Y, gamma NHLH1 NT_004982:183 NM_005598 686 bHLH nescient helix loop helix 1 NHLH2 NM_005599 687 bHLH nescient helix loop helix 2 NKX2-2 NM_002509 688 Homeobox NK2 transcription factor related, locus 2 (Drosophila) NKX2-3 NM_145285 689 Homeobox NK2 transcription factor related, locus 3 (Drosophila) NKX2-4 AF202037 690 Homeobox NK2 transcription factor related, locus 4 (Drosophila) NKX2-5 NM_004387 691 Homeobox NK2 transcription factor related, locus 5 (Drosophila) NKX2-8 NM_014360 692 Homeobox NK2 transcription factor related, locus 8 (Drosophila) NKX3-1 NM_006167 693 Homeobox NK3 transcription factor related, locus 1 (Drosophila) NKX6-1 NM_006168 694 Homeobox NK6 transcription factor related, locus 1 (Drosophila) NKX6-2 NM_177400 695 Homeobox NK6 transcription factor related, locus 2 (Drosophila) NM1 NM_004688 696 Co-activator N-myc (and STAT) interactor NPAS1 NM_002517 697 bHLH neuronal PAS domain protein 1 NR1D2 NM_005126 698 NHR subfamily 1, group D, member 2 NR1H2 NM_007121 699 NHR nuclear receptor subfamily 1, group H, member 2 NR1H3 NM_005693 700 NHR nuclear receptor subfamily 1, group H, member 3 NR1H4 NM_005123 701 NHR nuclear receptor subfamily 1, group H, member 4 NR1I2 NM_003889.3 702 NHR nuclear receptor subfamily NM_022002.2 1485 1, group I, member 2 NM_033013.2 1486 (isoforms 1-3) NRI13 NM_005122 703 NHR nuclear receptor subfamily 1, group I, member 3 NR2C1 NM_003297 704 NHR nuclear receptor subfamily 2, group C, member 1 NR2C2 NM_003298 705 NHR nuclear receptor subfamily 2, group C, member 2 NR2E1 NM_003269 706 NHR nuclear receptor subfamily 2, group E, member 1 NR2E3 NM_016346 707 NHR nuclear receptor subfamily 2, group E, member 3 NR2F1 NM_005654 708 NHR nuclear receptor subfamily 2, group F, member 1 NR2F2 NM_021005 709 NHR nuclear receptor subfamily 2, group F, member 2 NR2F6 NM_005234 710 NHR nuclear receptor subfamily 2, group F, member 6 NR3C1 NM_000176 711 NHR nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR3C2 NM_000901 712 NHR nuclear receptor subfamily 3, group C, member 2 NR4A1 NM_002135 713 NHR nuclear receptor subfamily 4, group A, member 1 NR4A2 NM_006186 714 NHR nuclear receptor subfamily 4, group A, member 2 NR4A3 NM_006981 715 NHR nuclear receptor subfamily 4, group A, member 3 NR5A1 NM_004959 716 NHR nuclear receptor subfamily 5, group A, member 1 NR5A2 NM_003822 717 NHR nuclear receptor subfamily 5, group A, member 2 NR6A1 NM_001489 718 NHR nuclear receptor subfamily 6, group A, member 1 NRF NM_017544 719 Other transcription factor OG2x AC004534 720 Homeobox homeobox (mouse) homolog OLIG1 BCO26989 721 bHLH oligodendrocyte transcription factor 1 OLIG2 NM_005806 722 bHLH oligodendrocyte transcription factor 2 OLIG3 NM_175747 723 bHLH oligodendrocyte transcription factor 3 ONECUT1 U96173 724 Homeobox one cut domain, family member 1 ONECUT2 NM-004852 725 Homeobox one cut domain, family member 2 OPTN NM_021980 726 Co-activator optineurin OSR1 NM_145260 727 ZnF-C2H2 odd-skipped related 1 OSR2 NT_008046:515 NM_053001 728 ZnF-C2H2 odd-skipped-related 2A protein OTEX NT_011588:87 NM_139282 729 Homeobox paired-like homeobox protein OTEX OTP NT_006713:546 NM_032109 730 Homeobox orthopedia homolog (Drosophila) OTX1 NM_014562 731 Homeobox orthodenticle homolog 1 (Drosophila) OTX2 NM_021728 732 Homeobox orthodenticle homolog 2 (Drosophila) OTX3 NM_147192 733 Homeobox orthodenticle homolog 3 (Drosophila) OVOL1 NM_004561 734 ZnF-C2H2 ovo-like 1 (Drosophila) OVOL3 AD001527 735 ZnF-C2H2 ovo-like 3 (Drosophila) p100 NM_014390 736 Co-activator EBNA-2 Co-activator (100 kD) P1P373C6 NM_019110 737 ZnF-C2H2 hypothetical protein P1 p373c6 P381IP NM_017569 738 Other transcription factor (p38 interacting protein) PAWR NT_019546:106 NM_002583 739 bZIP PRKC, apoptosis, WT1, regulator PAX1 NM_006192 740 Paired Box paired box gene 1 PAX2 NM_000278 741 Paired Box paired box gene 2 PAX3 NM_000438 742 Paired Box paired box gene 3 (Waardenburg syndrome 1) PAX4 NM_006193 743 Paired Box paired box gene 4 PAX5 NM_016734 744 Paired Box paired box gene 6 (B-cell lineage specific activator protein) PAX6 NM_000280 745 Paired Box paired box gene 6 (aniridia, keratitis) PAX7 NM_002584 746 Paired box paired box gene 7 PAX8 NM_003466 747 Paired Box paired box gene 8 PAX9 NM_006194 748 Paired Box paired box gene 9 PAXIP1L U80735 749 Co-activator PAX transcription activation domain interacting protein 1 like PBX1 NM_002585 750 Homeobox pre-B-cell leukemia transcription factor 1 PBX2 NM_002586 751 Homeobox pre-B-cell leukemia transcription factor 2 glutamine/Q-rich- associated protein PDEF NM_012391 752 Trp cluster- prostate epithelium- Ets specific Ets transcription factor PEGASUS NM_022466 753 ZnF-C2H2 zinc finger protein, subfamily 1A, 5 (Pegasus) PER1 NM_002616 754 bHLH period homolog 1 (Drosophila) PER2 NM_003894 755 bHLH period homolog 2 (Drosophila) PER3 NM_016831 756 bHLH period homolog 3 (Drosophila) PFDN5 NM_002624 757 Co-repressor prefoldin 5 PGR NM_000926 758 NHR progesterone receptor PHC1 NM_004426 759 Structural polyhomeotic-like 1 (Drosophila) PHD3 NM_015153 760 ZnF-PHD PHD finger protein 3 PHF15 NT_034776:94 NM_015288 761 ZnF-PHD PHD finger protein 15 PHF16 NT_011568:120 NM_014735 762 ZnF-PHD PHD finger protein 6 PHTF1 NM_006608 763 Homeobox putative homeodomain transcription factor PIAS1 NT_010222:2 NM_016166 764 ZnF-MIZ protein inhibitor of activated STAT, 1 PIAS3 NM_006099 765 ZnF-MIZ protein inhibitor of activated STAT3 PIASY NT_011255:153 NM_015897 766 ZnF-MIZ protein inhibitor of activated STAT protein PIASy PIG7 NM_004862 767 Other LPS-induced TNF-alpha factor PILB NM_012228 768 Other pilin-like transcription factor PITX1 NM_002653 769 Homeobox paired-like homeodomain transcription factor 1 PITX2 NM_000325 770 Homeobox paired-like homeodomain transcription factor 2 PITX3 NM_005029 771 Homeobox paired-like homeodomain transcription factor 3 PKNOX1 NM_004571 772 Homeobox PBX/knotted 1 homeobox 1 PKNOX2 NM_022062 773 Homeobox PBX/knotted 1 homeobox 2 PLAG1 NM_002655 774 ZnF-C2H2 pleiomorphic adenoma gene 1 PLGAL1 NM_002656 775 ZnF-C2H2 pleiomorphic adenoma gene-like 1 PLAGL2 NM_002657 776 ZnF-C2H2 pleiomorphic adenoma gene-like 2 PLRG1 NM_002669 777 Co-repressor pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PMF1 NM_007221 778 Co-activator polyamine-modulated factor 1 PML NM_002675 779 Structural promyelocytic leukemia PMX1 NM_006902 780 Homeobox paired mesoderm homeo box 1 POU3F1 NM_002699 781 Homeobox POU domain, class 3, transcription factor 1 POU3F2 NM_005604 782 Homeobox POU domain, class 3, transcription factor 2 POU3F3 NM_006236 783 Homeobox POU domain, class 3, transcription factor 3 POU3F4 NM_000307 784 Homeobox POU domain, class 3, transcription factor 4 POU4F1 NM_006237 785 Homeobox POU domain, class 4, transcription factor 1 POU4F2 NM_004575 786 Homeobox POU domain, class 4, transcription factor 2 POU4F3 NM_002700 787 Homeobox POU domain, class 4, transcription factor 3 POU5F1 NM_002701 788 Homeobox POU domain, class 5, (OCT4) transcription factor 1 POU6F1 NM_002702 789 Homeobox POU domain, class 6, transcription factor 1 PPARA NM_005036 790 NHR peroxisome proliferative activated receptor, alpha PPARBP NM_004774 791 Co-activator peroxisome proliferator activated receptor binding protein PPARD NM_006238 792 NHR peroxisome proliferative activated receptor, delta PPARG NM_005037 793 NHR peroxisome proliferative activated receptor, gamma PPARGC1 NM_013261 794 Co-activator peroxisome proliferative activated receptor, gamma, coactivator 1 PRDM1 NM_001198 795 Structural PR domain containing 1, with ZNF domain PRDM10 NM_020228 796 Structural PR domain containing 10 PRDM11 NM_020229 797 Structural PR domain containing 11 PRDM12 NM_021619 798 Structural PR domain containing 12 PRDM13 NM_021620 799 Structural PR domain containing 13 PRDM14 NM_024504 800 Structural PR domain containing 14 PRDM15 NM_144771 801 Structural PR domain containing 15 PRDM16 NM_022114 802 Structural PR domain containing 16 PRDM2 NM_012231 803 Structural PR domain containing 2, with ZNF domain PRDM4 NM_012406 804 Structural PR domain containing 4 PRDM5 NM_018699 805 Structural PR domain containing 5 PRDM6 AF272898 806 Structural PR domain containing 6 PRDM7 AF274348 807 Structural PR domain containing 7 PRDM8 NM_020226 808 Structural PR domain containing 8 PROX1 NM_002763.3 809 Homeobox homeobox 1 PRX2 NM_016307 810 Homeobox paired related homeobox protein PSIP1 NM_021144.3 811 Co-activator PC4 and SFRS1 NM_001128217.1 1487 interacting protein 1 NM_033222.3 1488 (isoforms 1-3) PSMC2 NT_007933:2739 NM_002803 812 Co-activator proteasome (prosome, macropain) 26S subunit, ATPase, 2 PSMC5 NM_002805 813 Co-activator proteasomes (prosome, macropain) 26S subunit, ATPase, 5 PTF1A NT_008705:1995 NM_178161 814 bHLH pancreas specific transcription factor, 1a PTTG1IP NM_004339 815 Co-activator pituitary tumor- transforming 1 interacting protein PURA NM_005859 816 Other purine-rich element binding protein A R28830_2 AC003682 817 ZnF-Other similar to ZNF197 (ZNF20) R32184_3 NM_033420 818 Other hypothetical protein MGC4022 RAI NM_006663 819 Co-repressor RelA-associated inhibitor RAI15 U50383 820 Other retinoic acid induced 15 RAA NM_000964 821 NHR retinoic acid receptor, alpha RARB NM_000965 822 NHR retinoic acid receptor, beta RARG NM_000966.4 823 NHR retinoic acid receptor, NM_001042728.1 1489 gamma (isoforms 1-2) RAX NM_013435 824 Homeobox retina and anterior neural fold homeobox RB1 NM_000321 825 Pocket retinoblastoma 1 domain (including osteosarcoma) RBAF600 AB007931 826 ZnF-C2H2 retinoblastoma-associated factor 600 RBAK NT_007819:532 NM_021163 827 Other RB-associated KRAB repressor RBBP5 NM_005057 828 Co-repressor retinoblastoma binding protein 5 RBBP9 NM_006606 829 Co-repressor retinoblastoma binding protein 9 RBL1 NM_002895 830 Pocket retinoblastoma-like 1 domain (p107) RBL2 NM_005611 831 Pocket retinoblastoma-like 2 domain (p130) RBPSUH NM_016270 832 ZnF-C2H2 recombining binding protein suppressor of hairless (Drosophila) RBPSUHL NM_014276 833 Other recombining binding protein suppressor of hairless-like (Drosophila) RCOR NM_015156 834 Other REST corepressor RCV1 NM_002903 835 Other recoverin REL NM_002908 836 Beta-scaffold- v-rel reticuloendotheliosis RHD viral oncogene (avian) REQ NM_006268 837 ZnF-PHD requiem, apoptosis response zinc finger gene RERE NM_012102 838 Other arginine-glutamic acid dipeptide (RE) repeats REST NM_005612 839 ZnF-C2H2 RE1-silencing transcription factor TRIM27 NT_033168:4 NM_006510.4 840 Structural tripartite motif containing 27 TRIM13 NM_005798.3 841 Structural tripartite motif containing NM_052811.2 1499 13 (isoforms 1, 1, 1, and 2, NM_213590.1 1500 respectively) NM_001007278.1 136 RFPL3 NT_011520:1735 NM_006604 842 Structural ret finger protein-like 3 RFX1 NM_002918 843 RFX regulatory factor X, 1 (influences HLA class II expression) RFX2 NM_000635 844 RFX regulatory factor X, 2 (influences HLA class II expression) RFX3 NM_002919 845 RFX regulatory factor X, 3 (influences HLA class II expression) RFX4 NM_002920 846 RFX regulatory factor X, 4 (influences HLA class II expression) RFX5 NM_000449 847 RFX regulatory factor X, 5 (influences HLA class II expression) RFXANK NM_003721 848 Co-activator regulatory factor X- associated ankyrin- containing protein RGC32 NM_014059 849 Other RGC32 protein RIN3 NT_026437:2459 NM_024832 850 bHLH Ras and Rab interactor 3 RING1 NM_002931 851 ZnF-Other ring finger protein 1 RIP60 NM_013400 852 ZnF-C2H2 replication initiation region protein (60 kD) RIPX NT_006216:11 NM_014961 853 ZnF-PHD rap2 interacting protein x RLF NM_012421 854 ZnF-C2H2 rearranged L-myc fusion sequence RNF10 NM_014868 855 ZnF-Other ring finger protein 10 RNF12 NM_016120 856 ZnF-Other ring finger protein 12 RNF13 NM_007282 857 ZnF-Other ring finger protein 13 RNF135 NT_035420:144 NM_032322 858 ZnF-MIZ ring finger protein 135 isoform 1 RNF137 NT_028310:82 NM_018073 859 Structural ring finger protein 137 RNF14 NM_004290 860 Co-activator ring finger protein 14 RNF144 NM_014746 861 ZnF-Other Ring finger protein 144 RNF18 NT_033240:76 NM_020358 862 Structural ring finger protein 18 RNF2 NM_007212 863 Co-repressor ring finger protein 2 RNF24 NM_007219 864 ZnF-Other ring finger protein 24 RNF3 NM_006315 865 ZnF-Other ring finger protein 3 RNF36 NT_0101942 NM_080745 866 Structural ring finger protein 36 RNF4 NM_002938 867 ZnF-Other ring finger protein 4 RNF8 NM_003958 868 ZnF-Other ring finger protein (C3HC4 type) 8 RORA NM_134261.2 869 — RAR-related orphan NM_134260.2 1490 receptor A (isoforms a-d) NM_002943.3 1491 NM_134262.2 1492 RORB NM_006914.3 1493 RAR-related orphan receptor B RORC NM_005060.3 1494 RAR-related orphan NM_001001523.1 1495 receptor C (isoforms a-b) RUNX1 NM_001754 870 scaffold- (acute myeloid leukemia RUNT 1; aml1 oncogene) RUNX2 NM_004348 871 Beta-scaffold- runt-related transcription RUNT factor 2 RUNX3 NM_004350 872 Beta-scaffold- runt-related transcription RUNT factor 3 RXRA NM_002957 873 NHR retinoid X receptor, alpha RXRB NM_021976 874 NHR retinoid X receptor, beta RXRG NM_006917 875 NHR retinoid X receptor, gamma RYBP NT_005526:6 NM_012234 876 Co-repressor RING1 and YY1 binding protein SAFB NM_002967 877 Other scaffold attachment factor B SALL1 NM_002968 878 ZnF-C2H2 sal-like 1 (Drosophila) SALL2 AB002358 879 ZnF-C2H2 sal-like 2 (Drosophila) SALL3 NM_171999 880 ZnF-C2H2 sal-like 3 (Drosophila) SALL4 NM_020436 881 ZnF-C2H2 similar to SALL1 (sal (Drosophila)-like SAP18 NM_005870 882 Co-repressor sin3-associated polypeptide, 18 kD SAP30 NM_003864 883 Co-repressor sin3-associated polypeptide, 30 kd) SART3 NM_014706 884 Co-activator squamous cell carcinoma antigen recognized by T cells 3 SATB1 NM_002971 885 Homeobox special AT-rich sequence binding protein 1 (binds to nuclear matrix/scaffold- associating DNAs) SATB2 NT_005037:10 NM_015265 886 Homeobox SATB family member 2 SBB103 NM_005785 887 ZnF-Other hypothetical SBB103 protein SBLF NM_006873 888 Other stoned B-like factor SBZF3 NT_031730:7 NM_020394 889 ZnF-C2H2 zinc finger protein SBZF3 SCA2 NM_002973 890 Other spinocerebellar ataxia 2 (Olivopontocerebellar ataxia 2, autosomal dominant, ataxin 2) SCAND1 NM_016558 891 Co-activator SCAN domain-containing 1 SCAND2 NM_022050 892 Co-activator SCAN domain-containing 2 SCMH1 NT_004852:374 NM_012236 893 Structural sex comb on midleg homolog 1 (Drosophila) SCML1 NM_006746 894 Structural sex comb on midleg-like 1 (Drosophila) SCML2 NM_006089 895 Structural sex comb on midleg-like 2 (Drosophila) SCML4 NT_033944:303 NM_198081 896 Trp Cluster- sex comb on midleg-like 4 Ets SETDB1 NM_012432 897 Structural [cut off] bifurcated 1 SF1 NM_004630 898 ZnF-Other splicing factor 1 SHARP NM_015001 899 Co-repressor SMART/HDAC1 associated repressor protein SHOX NM_000451.3 900 Homeobox short stature homeobox NM_006883.2 1496 (isoforms a-b) SHOX2 NM_003030 901 Homeobox short stature homeobox 2 SIAH1 NM_003031 902 Co-repressor seven in absentia homolog 1 (Drosophila) SIAH2 NM_005067 903 Co-repressor seven in absentia homolog 2 (Drosophila) SIM1 NM_005068 904 bHLH single-minded homolog 1 (Drosophila) SIM2 NM_005069 905 bHLH single-minded homolog 2 (Drosophila) SIN3B AB014600 906 Co-activator 5IN3 homolog B, transcriptional regulator (yeast) SIX1 NM_005982 907 Homeobox sine oculis homeobox homolog 1 (Drosophila) SIX2 NM_016932 908 Homeobox sine oculis homeobox homolog 2 (Drosophila) SIX3 NM_005413 909 Homeobox sine oculis homeobox homolog 3 (Drosophila) SIX4 NM_017420 910 Homeobox sine oculis homeobox homolog 4 (Drosophila) SIX5 X84813 911 Homeobox sine oculis homeobox homolog 5 (Drosophila) SIX6 NM_007374 912 Homeobox sine oculis homeobox homolog 6 (Drosophila) SLB AL110218 913 Co-repressor selective LIM binding factor SLC2A4RG NT_011333:173 NM_020062 914 ZnF-C2H2 SLC2A4 regulator SMARCA1 NM_003069 915 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1 SMARCA2 NM_003070 916 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 SMARCA3 NM_003071 917 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 3 SMARCA4 NM_003072 918 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 subfamily a-like 1 SMARCB1 NM_003073 919 Other SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 SMARCC1 NM_003074 920 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1 SMARCC2 NM_003075 921 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 2 SMARCE1 NM_003079 922 Structural SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1 KDM5C NM_004187.3 923 Structural lysine (K)-specific NM_001146702.1 924 demethylase 5C SNAI1 NM_005985 925 ZnF-C2H2 snail homolog 1 (Drosophila) SNAI2 NM_003068 926 ZnF-C2H2 snail homolog 2 (Drosophila) SNAI3 BC041461 927 ZnF-C2H2 snail homolog 3 (Drosophila) SNAPC1 NM_003082 928 Other small nuclear RNA activating complex, polypeptide 1, 43 kDa SNAPC2 NM_003083 929 Other small nuclear RNA activating complex, polypeptide 2, 45 kDa SNAPC3 NM_003084 930 Other small nuclear RNA activating complex, polypeptide 3, 50 kDa SNAPC4 NM_003086 931 Other small nuclear RNA activating complex, polypeptide 4, 190 kDa SNAPC5 NM_006049 932 Other small nuclear RNA activating complex, polypeptide 5, 19 kDa SNFT NM_018664 933 bZIP Jun dimerization protein p21SNFT SNW1 NM_012245 934 Co-activator SKI-interacting protein SOLH NT_010552:127 NM_005632 935 ZnF-PHD small optic lobes homolog (Drosophila) SOM NT_004391:39 NM_021180 936 Beta-scaffold- sister of mammalian grainyhead grainyhead SOX1 NM_005986 937 Beta-scaffold- SRY (sex determining HMG region Y)-box 1 SOX10 NM_006941 938 Beta-scaffold- SRY (sex determining HMG region Y)-box 10 SOX11 NM_003108 939 Beta-scaffold- SRY (sex determining HMG region Y)-box 11 SOX18 NM_018419.2 940 Beta-scaffold- SRY (sex determining HMG region Y)-box 18 SOX2 L07335 941 Beta-scaffold- (sex determining region HMG Seed Y)-box 2 SRY SOX21 NM_007084 942 Beta-Scaffold- SRY (Sex determining HMG region Y)-box 21 SOX3 NM_005634 943 Beta-Scaffold- SRY (sex determining HMG region Y)-box 3 SOX30 NM_007017 944 Beta-scaffold- SRY (sex determining HMG region Y)-box 30 SOX4 NM_003107 6659 945 Beta-scaffold- SRY (sex determining HMG region Y)-box 4 SOX5 NM_006940 6660 946 Beta-scaffold- SRY (sex determining HMG region Y)-box 5 SOX6 NM_033326 947 Beta-scaffold- SRY (sex determining 55553 HMG region Y)-box 6 SOX7 NT_008010:24 NM_031439 948 Beta-scaffold- SRY (sex determining HMG region Y)-box 7 SOX8 NM_014587 949 Beta-scaffold- SRY (sex determining 30812 HMG region Y)-box 8 SOX9 NM_000346 6662 950 Beta-scaffold- SRY (sex determining HMG region Y)-box 9 SP1 J03133 951 ZnF-C2H2 Sp1 transcription factor SP100 NT_005403:864 NM_003113 952 Beta-scaffold- nuclear antigen Sp100 HMG SP2 NM_003110 953 ZnF-C2H2 Sp2 transcription factor SP3 X68560 954 ZnF-C2H2 Sp3 transcription factor SP4 NM_003112 955 ZnF-C2H2 Sp4 transcription factor SP7 NT_009563:27 NM_152860 956 ZnF-C2H2 Sp7 transcription factor SPI1 NM_003120 957 Trp cluster- spleen focus forming virus Ets (SFFV) proviral integration oncogene spi1 SPIB NM_003121 958 Trp cluster- Spi-B transcription factor Ets (Spi-1/PU.1 related) SPIC NT_009743:37 NM_152323 959 Trp Cluster- likely ortholog of mouse Ets Spi-C transcription factor (Spi-1/PU.1 related) SRA1 AF293024 960 Co-activator steroid receptor RNA activator 1 SRCAP NM_006662 961 Structural Snf2-related CBP activator protein SREBF1 NM_004176 962 bHLH sterol regulatory element binding transcription factor 1 SREBF2 NM_004599 963 bHLH sterol regulatory element binding transcription factor 2 SRF NM_003131 964 Beta-scaffold- serum response factor (c- MADS fos serum response element-binding transcription factor) SRY NM_003140 965 Beta-scaffold- sex determining region Y HMG SSA1 NT_028310:75 NM_003141 966 Structural Sjogren syndrome antigen A1 (52 kDa, ribonucleoprotein autoantigen SS-A/Ro) SSRP1 NM_003146 967 Co-activator structure specific recognition protein 1 SSX1 NM_005635 968 Other synovial sarcoma, X breakpoint 1 SSX2 NM_003147 969 Other synovial sarcoma, X breakpoint 2 SSX3 NM_021014 970 Other synovial sarcoma, X breakpoint 3 SSX4 NM_005636 971 Other synovial sarcoma, X breakpoint 4 SSX5 NM_021015 972 Other synovial sarcoma, X breakpoint 5 SSX6 NM_173357 973 Other synovial sarcoma, X breakpoint 6 SSX7 NM_173358 974 Other synovial sarcoma, X breakpoint 7 SSX8 NM_174961 975 Other synovial sarcoma, X breakpoint 8 SSX9 NM_174962 976 Other synovial sarcoma, X breakpoint 9 ST18 NM_014682 977 ZnF-C3H suppression of tumorigenicity 18 (breast carcinoma) (zinc finger protein) STAT1 NM_007315 978 Beta-scaffold- signal transducer and STAT activator of transcription 1, 91 kDa STAT2 NM_005419 979 Beta-scaffold- signal transducer and STAT activator of transcription 2, 113 kDa STAT3 NM_003150 980 Beta-scaffold- signal transducer and STAT activator of transcription 3 (acute-phase response factor) STAT4 NM_003151 981 Beta-scaffold- signal transducer and STAT activator of transcription 4 STAT5A NM_003152 982 Beta-scaffold- signal transducer and STAT activator of transcription 5A STAT5B NM_012448 983 Beta-scaffold- signal transducer and STAT activator of transcription 5B STAT6 NM_003153 984 Beta-scaffold- signal transducer and STAT activator of transcription 6, interleukin-4 induced SUPT16H NM_007192 985 Other suppressor of Ty 16 homolog (S. cerevisiae) SUPT3H NM_003599 986 Other suppressor of Ty 3 homolog (S. cerevisiae) SUPT4H1 NM_003168 987 Other suppressor of Ty 4 homolog (S. cerevisiae) SUPT5H NM_003169 988 Dwarfin suppressor of Ty 5 homolog (S. cerevisiae) SUPT6H NM_003170 989 Other suppressor of Ty 6 homolog (S. cerevisiae) SURB7 NM_004264 990 Other SRB7 suppressor of RNA polymerase B homolog (yeast) SUV39H1 NT_011568:277 NM_003173 991 Structural suppressor of variegation 3-9 homolog 1 (Drosophila) SZF1: NT_022567:166 NM_016089 992 ZnF-C2H2 KRAB-zinc finger protein SZF1-1 SZFP41 NT_011192:184 NM_152279 993 ZnF-C2H2 zinc finger protein 41-like T NM_003181 994 T-box T, brachyury homolog (mouse) TADA2L NM_001488 995 Other transcriptional adaptor 2 (ADA2 homolog, yeast)- like TADA3L NM_006354 996 Other transcriptional adaptor 3 (ADA3 homolog, yeast)- like TAF1 NM_004606 997 Other TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250 kDa TAF10 NM_006284 998 Other TAF10 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 30 kDa TAF11 NM_005643 999 Other TAF11 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 28 kDa TAF12 NM_005644 1000 Other TAF12 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 20 kDa TAF13 NM_005645 1001 Other TAF13 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 18 kDa TAF15 NM_003487 1002 Other TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68 kDa TAF1A NM_005681 1003 Other TATA box binding protein (TBP)-associated factor, RNA polymerase I, A, 48 kDa TAF1B L39061 1004 Other TATA box binding protein (TBP)-associated factor, RNA polymemse 1, B, 63 kDa TAF1C NM_005679 1005 Other TATA box binding protein (TBP)-associated factor, RNA polymerase I, C, 110 kDa TAF2 NM_003184 1006 Other TAF2 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 150 kDa TAF3 AJ292190 1007 Other TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 140 kDa TAF4 NM_003185 1008 Other TAF4 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 135 kDa TAF4B Y09321 1009 Other TAF4b RNA polymerase II, TATA box binding protein (TBP)-associated factor, 80 kDa TAF6L NM_006473 1010 Co-activator TAF6-like RNA polymerase II, p300/CBP- associated factor (PCAF)- associated factor, 65 kDa TAF7 NM_005642 1011 Other TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 55 kDa TAF9 NM_003187 1012 Other TAF9 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 32 kDa TAL1 NM_003189 1013 bHLH T-cell acute lymphocytic leukemia 1 TAL2 NM_005421 1014 bHLH T-cell acute lymphocytic leukemia 2 TBP NM_003194 1015 Other TATA box binding protein TBPL1 NM_004865 1016 Other TBP-like 1 TBR1 NM_006593 1017 T-box T-box, brain, 1 TBX1 NM_005992 1018 T-box T-box 1 TBX10 AF033579 1019 T-box T-box 10 TBX15 NM_152380 1020 T-box T-box 15 TBX18 AJ010278 1021 T-box T-box 18 TBX19 NM_005149 1022 T-box T-box 19 TBX2 NM_005994 1023 T-box T-box 2 TBX20 AJ237589 1024 T-box T-box 20 TBX21 NM_013351 1025 T-box T-box 21 TBX22 NM_016954 1026 T-box T-box 22 TBX3 NM_005996 1027 T-box T-box 3 (ulnar mammary syndrome) TBX4 NM_018488 1028 T-box T-box 4 TBX5 NM_000192 1029 T-box T-box 5 TBX6 NM_004608 1030 T-box T-box 6 TCEAL1 NM_004780 1031 ZnF-Other transcription elongation factor A (SII)-like 1 TCERG1 NM_006706 1032 Other transcription elongation regulator 1 (CA150) TCF1 NM_000545 1033 Homeobox transcription factor 1, hepatic; LF-B1, hepatic nuclear factor (HNF1), albumin proximal factor TCF12 NM_003205 1034 bHLH transcription factor 12 (HTF4, helix-loop-helix transcription factors 4) TCF15 NM_004609 1035 bHLH transcription factor 15 (basic helix-loop-helix) TCF19 NM_007109 1036 Other transcription factor 19 (SC1) TCF7 NM_003202 1037 scaffold- transcription factor 2, (T- HMG cell specific, HMG-box) TCF7L1 X62870 1038 Beta-scaffold- transcription factor 7-like HMG 1 (T-cell specific, HMG- box) TCF7L2 NM_030756 1039 Beta-scaffold- transcription factor 7-like HMG 2 (T-cell specific, HMG- box) TCF8 NM_030751 1040 ZnF-C2H2 transcription factor 8 (represses interleukin 2 expression) TCFL1 NM_005997 1041 Other transcription factor-like 1 TCFL4 NM_013383 1042 bHLH transcription factor-like 4 TCFL5 NM_006602 1043 bHLH transcription factor-like 5 (basic helix-loop-helix) TEAD1 NM_021961 1044 TEA TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD2 NM_003598 1045 TEA TEA domain family member 2 TEAD3 NM_003214 1046 TEA TEA domain family member 3 TEAD4 NM_003213 1047 TEA TEA domain family member 4 TEF NM_003216 1048 bZIP thyrotrophic embryonic factor TEL2 NM_016135 1049 Trp cluster- ets transcription factor Ets TEL2 TEX27 NM_021943 1050 ZnF-AN1 testis expressed sequence 27 TFAM NM_012251 1051 Beta-scaffold- transcription factor A, HMG mitochondrial TFAP2A NM_003220 1052 AP-2 transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) TFAP2B NM_003221 1053 AP-2 transcription factor AP-2 beta (activating enhancer binding protein 2 beta) TFAP2BL1 NM_172238 1054 AP-2 transcription factor AP-2 beta (activating enhancer binding protein 2 beta)- like 1 TFAP2C NM_003222 1055 AP-2 transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) TFAP4 NM_003223 1056 bHLH transcription factor AP-4 (activating enhancer binding protein 4) TFB1M NM_016020 1057 Other transcription factor Bl, mitochondrial TFB2M NM_022366 1058 Other transcription factor B2, mitochondrial TFCP2 NM_005653 1059 Beta-scaffold- transcription factor CP2 grainyhead TFE3 NM_006521 1060 bHLH transcription factor binding to IGHM enhancer 3 TFEB BC006225 1061 bHLH transcription factor EB TFEC NM_012252 1062 bHLH transcription factor EC TGFB1I1 NM_015927 1063 Co-activator transforming growth factor beta 1 induced transcript 1 TGIF NM_003244 1064 Homeobox TGFB-induced factor (TALE family homeobox) THG-1 AJ133115 1065 bZIP TSC-22-like THRA NM_003250 1066 NHR thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) THRAP4 NM_014815 1067 Co-activator thyroid hormone receptor associated protein 4 THRB NM_000461 1068 NHR thyroid hormone receptor, beta (erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian) TIEG NM_005655 1069 ZnF-C2H2 TGFB inducible early growth response TIEG2 NM_003597 1070 ZnF-C2H2 TGFB inducible early growth response 2 TIF11 NM_003852 1071 Structural transcriptional intermediary factor 1 TIMELESS NM_003920 1072 Other timeless homolog (Drosophila) TIP120A NM_018448 1073 Co-activator TBP-interacting protein TITF1 NM_003317 1074 Homeobox thyroid transcription factor 1 TIX1 AB007855 1075 Homeobox triple homeobox 1 TIZ NT_033317:106 NM_138330 1076 ZnF-C2H2 TRAF6-inhibitory zinc finger protein TLX1 NM_005521 1077 Homeobox T-cell leukemia, homeobox 1 TLX2 NM_001534 1078 Homeobox T-cell leukemia, homeobox 2 TLX3 NM_021025 1079 Homeobox T-cell leukemia, homeobox 3 TMF1 NM_007114 1080 Other TATA element modulatory factor 1 TNRC11 NM_005120 1081 Co-activator trinucleotide repeat containing 11 (THR- associated protein, 230 kDa subunit) TNRC17 U80752.1 1082 Other trinucleotide repeat containing 17 TNRC18 U80753 1083 Other trinucleotide repeat containing 18 TNRC21 U80756 1084 Other trinucleotide repeat containing 21 TNRC3 NM_005878 1085 Other trinucleotide repeat containing 3 TP53 NM_000546 1086 Beta-scaffold- tumor protein P53 (Li- p53 Fraumeni syndrome) TP53BP2 NT_004525:42 NM_005426 1087 Co-repressor tumor protein p53 binding protein, 2 TP63 NM_003722 1088 Beta-scaffold- tumor protein p63 p53 TP73 NM_005427 1089 Beta-scaffold- tumor protein p73 p53 TRAP150 NM_005119 1090 Co-activator thyroid hormone receptor- associated protein, 150 kDa subunit TRAP95 NM_005481 1091 Co-activator thyroid hormone receptor- associated protein, 95-kD subunit TRERF1 NT_007592:3400 NM_018415 1092 ZnF-C2H2 transcriptional regulating factor 1 TRIM10 NM_006778 1093 Structural tripartite motif-containing 10 TRIM14 NT_033216:170 NM_014788 1094 Structural tripartite motif-containing 14 TRIM15 NM_033229 1095 Structural tripartite motif-containing 15 TRIM16 NT_010718:517 NM_006470 1096 Structural tripartite motif-containing 16 TRIM17 NT_004908:93 NM_016102 1097 Structural tripartite motif-containing 17 TRIM22 NM_006074 1098 Structural tripartite motif-containing 22 TRIM26 NM_003449 1099 Structural tripartite motif-containing 26 TRIM28 NM_005762 1100 Structural tripartite motif-containing 28 TRIM29 NT_033899:65 NM_012101 1101 Structural tripartite motif-containing 29 TRIM3 NM_006458 1102 ZnF-Other tripartite motif-containing 3 TRIM31 NT_034873:26 NM_007028 1103 Structural tripartite motif-containing 31 TRIM33 NM_015906 1104 Structural tripartite motif-containing 33 TRIM34 NT_03508:27a NM_021616 1105 Structural tripartite motif-containing 34 TRIM35 NT_007988:5 NM_015066 1106 Structural tripartite motif-containing 35 TRIM38 NM_006355 1107 ZnF-Other tripartite motif-containing 38 TRIM39 NT_033951:12 NM_021253 1108 Structural tripartite motif-containing 39 TRIM4 NT_007933:2024 NM_033017 1109 Structural tripartite motif-containing 4 TRIM40 NT_007592:1918 NM_138700 1110 Structural tripartite motif-containing 40 TRIM41 NT_006519:206 NM_201627 1111 Structural tripartite motif-containing 41 TRIM47 NT_033292:11 NM_033452 1112 Structural tripartite motif-containing 47 TRIM48 NT_033903:1 NM_024114 1113 Structural tripartite motif-containing 48 TRIM5 NT_035080:27b NM_033034 1114 Structural tripartite motif-containing 5 TRIP11 NM_004239 1115 Co-activator thyroid hormone receptor interactor 11 TRIP11 NM_004237 1116 Co-activator thyroid hormone receptor interactor 13 TRIP15 NM_004236 1117 Co-activator thyroid receptor interacting protein 15 TRIP4 NM_016213 1118 Co-activator thyroid hormone receptor interactor 4 TRIP6 L40374 1119 Co-activator thyroid hormone receptor interactor 6 TRIP8 NT_008583:38 NM_004241 1120 Jumonji thyroid hormone receptor interactor 8 TRIP-Br2 NM_014755 1121 Co-activator transcriptional regulator interacting with the PHS- bromodomain 2 TRPS1 NM_014112 1122 ZnF -Other trichorhinophalangeal syndrome I TSC22 NM_006022 1123 bZIP transforming growth factor beta-stimulated protein TSC-22 TUB NM_003320 1124 Tubby tubby homolog (mouse) TULP1 NM_003322 1125 Tubby tubby like protein 1 TULP2 NM_003323 1126 Tubby tubby like protein 2 TULP3 NM_003324 1127 Tubby tubby like protein 3 TULP4 NM_020245 1128 Tubby tubby like protein 4 TWIST NM_000474 1129 bHLH Twist TZFP NM_014383 1130 ZnF- testis zinc finger protein BTB/POZ UBP1 NM_014517 1131 Beta-scaffold- upstream binding protein 1 grainyhead (LBP-1a) UBTF NM_014233 1132 Beta-scaffold- upstream binding HMG transcription factor, RNA polymerase 1 UHRF1 NM_013282 1133 ZnF-PHD ubiquitin-like, containing PHD and RING finger URF2 NT_008413:704 NM_152306 1134 ZnF-PHD ubiquitin-like, containing PHD and RING finger domains 2 USF1 NM_007122 1135 bHLH upstream transcription factor 1 USF2 NM_003367 1136 bHLH upstream transcription factor 2, c-fos interacting UTF1 NM_003577 1137 bZIP undifferentiated embryonic cell transcription factor 1 VAX1 NM_199131 1138 Homeobox ventral anterior homeobox 1 VAX2 NM_012476 1139 Homeobox ventral anterior homeobox 2 VDR NM_000376 1140 NHR vitamin D (1, 25- dihydroxyvitamin D3) receptor VENTX2 NM_014468 1141 Homeobox VENT-like homeobox 2 VIK NT_007933:1990 NM_024061 1142 ZnF-C2H2 vav-1 interacting Kruppel- like protein cutoff YAF2 NM_005748 1143 Co-repressor YY1 associated factor 2 YBX2 NM_015982 1144 Beta-scaffold- germ cell specific Y-box cold-shock binding protein YY1 NM_003403 1145 ZnF-C2H2 YY1 transcription factor ZAR1 NM_175619 1146 Other zygote arrest 1 ZBTB1 NT_025892:3338 BC050719 1147 ZnF- zinc finger and BTB BTB/POZ domain containing 1 ZBTB2 NT_023451:235 NM_020861 1148 ZnF- zinc finger and BTB BTB/POZ domain containing 2 ZBTB4 NT_035416:6 NM_020899 1149 ZnF-C2H2 zinc finger and BTB domain containing 4 ZDHHC1 U90653 1150 ZnF-Other zinc finger, DHHC domain containing 1 ZF NM_021212 1151 bZIP HCF-binding transcription factor Zhangfei ZF5128 NM_014347 1152 ZnF-C2H2 zinc finger protein ZFD25 NM_016220 1153 ZnF-C2H2 zinc finger protein (ZFD25) ZFH4 NT_008055:104 NM_024721 1154 ZnF-C2H2 zinc finger homeodomain 4 ZFHX1B NM_014795 1155 ZnF-C2H2 zinc finger homeobox 1B ZFHX2 AB051549 1156 Homeobox zinc finger homeobox 2 ZFP NM_018651 1157 ZnF-C2H2 zinc finger protein ZFP1 NT_035368:196 NM_153688 1158 ZnF-C2H2 zinc finger protein homolog ZFP100 AL080143 1159 ZnF-C2H2 zinc finger protein ZFP103 NM_005677 1160 ZnF-Other zinc finger protein 103 homolog (mouse) ZFP106 NM_022473 1161 ZnF-C2H2 zinc finger protein 106 ZFP161 NM_003409 1162 ZnF- zinc finger protein 161 BTB/POZ homolog (mouse) ZFP26 NM_016422 1163 ZnF-Other C3HC4-like zinc finger protein ZFP276 NT_010542:164 NM_152287 1164 ZnF-C2H2 zinc finger protein 276 homolog ZFP28 AB037852 1165 ZnF-C2H2 zinc finger protein 28 homolog (mouse) ZFP289 NM_032389 1166 ZnF-Other Seed zinc finger protein 289, ID1 regulated ZFP29 NM_017894 1167 ZnF-C2H2 likely ortholog of mouse zinc finger protein 29 ZFP318 NM_014345 1168 ZnF-Other Seed endocrine regulator ZFP36 NM_003407 1169 ZnF-C3H zinc finger protein 36, C3H type, homolog (mouse) ZFP37 NM_003408 1170 ZnF-C2H2 zinc finger protein 37 homolog (mouse) ZFP42 NT_022841:73 NM_174900 1171 ZnF-C2H2 Found zinc finger protein 42 ZFP64 NM_018197 1172 ZnF-C2H2 Seed zinc finger protein 64 homolog (mouse) ZFP67 NM_015872 1173 ZnF- Seed zinc finger protein 67 BTB/POZ homolog (mouse) ZFP91 AB056107 1174 ZnF-C2H2 zinc finger protein 91 homolog (mouse) ZFP92 U82695 1175 ZnF-Other zinc finger protein 92 homolog (mouse) ZFP95 NM_014569 1176 ZnF-C2H2 zinc finger protein 95 homolog (mouse) ZFPL1 NM_006782 1177 ZnF-PHD zinc finger protein-like 1 ZFPM1 NM_153813 1178 ZnF-C2H2 zinc finger protein, multitype 1 (FOG1) ZFPM2 NM_012082 1179 ZnF-C2H2 zinc finger protein, multitype 2 (FOG2) ZFR NM_016107 1180 ZnF-C2H2 zinc finger RNA binding protein ZFX NM_003410 1181 ZnF-C2H2 zinc finger protein, X-linked ZFY NM_003411 1182 ZnF-C2H2 zinc finger protein, Y-linked ZHX1 NM_007222 1183 Homeobox zinc-fingers and homeoboxes 1 ZHX2 NT_023663:37 NM_014943 1184 Homeobox zinc fingers and homeoboxes 2 ZIC1 NM_003412 1185 ZnF-C2H2 Zic family member 1 (odd-paired homolog, Drosophila) ZIC2 NM_007129 1186 ZnF-C2H2 Zic family member 2 (odd-paired homolog, Drosophila) ZIC3 NM_003413 1187 ZnF-C2H2 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) ZIC4 NM_032153 1188 ZnF-C2H2 zinc finger protein of the cerebellum 4 ZIC5 NM_033132 1189 ZnF-C2H2 zinc finger protein of the cerebellum 5 ZID NM_006626 1190 ZnF- zinc finger protein with BTB/POZ interaction domain ZIM2 NM_015363 1191 ZnF-C2H2 zinc finger, imprinted 2 ZIM3 NT_011104:125 NM_052882 1192 ZnF-C2H2 zinc finger, imprinted 3 ZNF10 NM_003419 1193 ZnF-C2H2 zinc finger protein 10 (KOX 1) ZNF100 NT_035560:167 NM_173531 1194 ZnF-C2H2 zinc finger protein 100 ZNF117 NM_024498 1195 ZnF-C2H2 zinc finger protein 117 (HPF9) ZNF11A X68686 1196 ZnF-C2H2 zinc finger protein ha (KOX 2) ZNF11B X68684 1197 ZnF-C2H2 zinc finger protein lib (KOX 2) ZNF123 S52506 1198 ZnF-C2H2 zinc finger protein 123 (HZF-1) ZNF124 NM_003431 1199 ZnF-C2H2 zinc finger protein 124 (HZF-16) ZNF125 S52508 1200 ZnF-C2H2 zinc finger protein 125 (HZF-3) ZNF126 S52507 1201 ZnF-C2H2 zinc finger protein 126 (HZF-2) ZNF131 U09410 1202 ZnF-C2H2 zinc finger protein 131 (clone pHZ-10) ZNF132 NM_003433 1203 ZnF-C2H2 zinc finger protein 132 (clone pHZ-12) ZNF133 NM_003434 1204 ZnF-C2H2 zinc finger protein 133 (clone pHZ-13) ZNF134 NM_003435 1205 ZnF-C2H2 zinc finger protein 134 (clone pHZ-15) ZNF135 NM_003436 1206 ZnF-C2H2 zinc finger protein 135 (clone pHZ-17) ZNF136 NM_003437 1207 ZnF-C2H2 zinc finger protein 136 (clone pHZ-20) ZNF137 NM_003438 1208 ZnF-C2H2 zinc finger protein 137 (clone pHZ-30) ZNF138 U09847 1209 ZnF-C2H2 zinc finger protein 138 (clone pHZ-32) ZNF14 NM_021030 1210 ZnF-C2H2 zinc finger protein 14 (KOX 6) ZNF140 NM_003440 1211 ZnF-C2H2 zinc finger protein 140 (clone pHZ-39) ZNF141 NM_003441 1212 ZnF-C2H2 zinc finger protein 141 (clone pHZ-44) ZNF142 NM_005081 1213 ZnF-C2H2 zinc finger protein 142 (clone pHZ-49) ZNF143 NM_003442 1214 ZnF-C2H2 zinc finger protein 143 (clone pHZ-1) ZNF144 NM_007144 1215 ZnF-Other zinc finger protein 144 (Mel-18) ZNF145 NM_006006 1216 ZnF- zinc finger protein 145 BTB/POZ (Kruppel-like, expressed in promyelocytic leukemia) ZNF146 NM_007145 1217 ZnF-C2H2 zinc finger protein 146 ZNF147 NM_005082 1218 Structural zinc finger protein 147 (estrogen-responsive finger protein) ZNF148 NM_021964 1219 ZnF-C2H2 zinc finger protein 148 (pHZ-52) ZNF151 NM_003443 1220 ZnF- zinc finger protein 151 BTB/POZ (pHZ-67) ZNF154 U20648 1221 ZnF-C2H2 zinc finger protein 154 (pHZ-92) ZNF155 NM_003445 1222 ZnF-C2H2 zinc finger protein 155 (pHZ-96) ZNF157 NM_003446 1223 ZnF-C2H2 zinc finger protein 157 (HZF22) ZNF15L1 X52339 1224 ZnF-C2H2 zinc finger protein 15-like 1 (KOX 8) ZNF16 NM_006958 1225 ZnF-C2H2 zinc finger protein 16 (KOX 9) ZNF160 X78928 1226 ZnF-C2H2 zinc finger protein 160 ZNF161 NM_007146 1227 ZnF-C2H2 zinc finger protein 161 ZNF165 NM_003447 1228 ZnF-C2H2 zinc finger protein 165 ZNF169 U28251 1229 ZnF-C2H2 zinc finger protein 169 ZNF17 AB075827 1230 ZnF-C2H2 zinc finger protein 17 (HPF3, KOX 10) ZNF174 NM_003450 1231 ZnF-C2H2 zinc finger protein 174 ZNF175 NM_007147 1232 ZnF-C2H2 zinc finger protein 175 ZNF177 NM_003451 1233 ZnF-C2H2 zinc finger protein 177 ZNF179 NM_007148 1234 ZnF-Other zinc finger protein 179 ZNF18 X52342 1235 ZnF-C2H2 zinc finger protein 18 (KOX 11) ZNF180 NM_013256 1236 ZnF-C2H2 zinc finger protein 180 (HHZ168) ZNF183 NM_006978 1237 ZnF-Other zinc finger protein 183 (RING finger, C3HC4 type) ZNF183L1 NT_009952:601 NM_178861 1238 ZnF-C3H zinc finger protein 183- like 1 ZNF184 U66561 1239 ZnF-C2H2 zinc finger protein 184 (Kruppel-like) ZNF185 NM_007150 1240 Co-activator zinc finger protein 185 (LIM domain) ZNF187 Z11773 1241 ZnF-C2H2 zinc finger protein 187 ZNF189 NM_003452 1242 ZnF-C2H2 zinc finger protein 189 ZNF19 NM_006961 1243 ZnF-C2H2 zinc finger protein 19 (KOX 12) ZNF192 NM_006298 1244 ZnF-C2H2 zinc finger protein 192 ZNF193 NM_006299 1245 ZnF-C2H2 zinc finger protein 193 ZNF195 NM_007152 1246 ZnF-C2H2 zinc finger protein 195 ZNF197 NM_006991 1247 ZnF-C2H2 zinc finger protein 197 ZNF2 Z60152 1248 ZnF-C2H2 zinc finger protein 2 (Al-5) ZNF20 AL080125 1249 ZnF-C2H2 zinc finger protein 20 (KOX 13) ZNF200 NM_003454 1250 ZnF-C2H2 zinc finger protein 200 ZNF202 NM_003455 1251 ZnF-C2H2 zinc finger protein 202 ZNF205 NM_003456 1252 ZnF-C2H2 zinc finger protein 205 ZNF207 NM_003457 1253 ZnF-C2H2 zinc finger protein 207 ZNF208 NM_007153 1254 ZnF-C2H2 zinc finger protein 208 ZNF21 X52345 1255 ZnF-C2H2 zinc finger protein 21 (KOX 14) ZNF211 NM_006385 1256 ZnF-C2H2 zinc finger protein 211 ZNF212 NM_012256 1257 ZnF-C2H2 zinc finger protein 212 ZNF213 AF017433 1258 ZnF-C2H2 zinc finger protein 213 ZNF214 NM_013249 1259 ZnF-C2H2 zinc finger protein 214 ZNF215 NM_013250 1260 ZnF-C2H2 zinc finger protein 215 ZNF216 NM_006007 1261 ZnF-AN1 zinc finger protein 216 ZNF217 NM_006526 1262 ZnF-C2H2 zinc finger protein 217 ZNF219 NM_016423 1263 ZnF-C2H2 zinc finger protein 219 ZNF22 NM_006963 1264 ZnF-C2H2 zinc finger protein 22 (KOX 15) ZNF220 NM_006766 1265 ZnF-PHD zinc finger protein 220 ZNF221 NM_013359 1266 ZnF-C2H2 zinc finger protein 221 ZNF222 NM_013360 1267 ZnF-C2H2 zinc finger protein 222 ZNF223 NM_013361 1268 ZnF-C2H2 zinc finger protein 223 ZNF224 NM_013398 1269 ZnF-C2H2 zinc finger protein 224 ZNF225 NM_013362 1270 ZnF-C2H2 zinc finger protein 225 ZNF226 NM_016444 1271 ZnF-C2H2 zinc finger protein 226 ZNF228 NM_013380 1272 ZnF-C2H2 zinc finger protein 228 ZNF229 AF192979 1273 ZnF-C2H2 zinc finger protein 229 ZNF23 AL080123 1274 ZnF-C2H2 zinc finger protein 23 (KOX 16) ZNF230 NM_006300 1275 ZnF-C2H2 zinc finger protein 230 ZNF232 NM_014519 1276 ZnF-C2H2 zinc finger protein 232 ZNF233 NT_011109:135 NM_181756 1277 ZnF-C2H2 zinc finger protein 233 ZNF234 X78927 1278 ZnF-C2H2 zinc finger protein 234 ZNF235 NM_004234 1279 ZnF-C2H2 zinc finger protein 235 ZNF236 NM_007345 1280 ZnF-C2H2 zinc finger protein 236 ZNF237 NM_014242 1281 ZnF-Other zinc finger protein 237 ZNF238 NM_006352 1282 ZnF-C2H2 zinc finger protein 238 ZNF239 NM_005674 1283 ZnF-C2H2 zinc finger protein 239 ZNF24 NM_006965 1284 ZnF-C2H2 zinc finger protein 24 (KOX 17) ZNF25 X52350 1285 ZnF-C2H2 zinc finger protein 25 (KOX 19) ZNF253 NT_011295:613 NM_021047 1286 ZnF-C2H2 zinc finger protein 253 ZNF254 NM_004876 1287 ZnF-C2H2 zinc finger protein 254 ZNF255 NM_005774 1288 ZnF-C2H2 zinc finger protein 255 ZNF256 NM_005773 1289 ZnF-C2H2 zinc finger protein 256 ZNF257 NT_033317:9 NM_033468 1290 ZnF-C2H2 zinc finger protein 257 ZNF258 NM_007167 1291 ZnF-Other zinc finger protein 258 ZNF259 NM_003904 1292 ZnF-Other zinc finger protein 259 ZNF26 NM_019591 1293 ZnF-C2H2 zinc finger protein 26 (KOX 20) ZNF261 NM_005096 1294 ZnF-Other zinc finger protein 261 ZNF262 NM_005095 1295 ZnF-Other zinc finger protein 262 ZNF263 NM_005741 1296 ZnF-C2H2 zinc finger protein 263 ZNF264 NM_003417 1297 ZnF-C2H2 zinc finger protein 264 ZNF265 NM_005455 1298 ZnF-Other zinc finger protein 265 ZNF266 X78924 1299 ZnF-C2H2 zinc finger protein 266 ZNF267 NM_003414 1300 ZnF-C2H2 zinc finger protein 267 ZNF268 AF317549 1301 ZnF-C2H2 zinc finger protein 268 ZNF271 NM_006629 1302 ZnF-C2H2 zinc finger protein 271 ZNF272 X78931 1303 ZnF-C2H2 zinc finger protein 272 ZNF273 X78932 1304 ZnF-C2H2 zinc finger protein 273 ZNF274 NM_016324 1305 ZnF-C2H2 zinc finger protein 274 ZNF275 NM_020636 1306 ZnF-C2H2 zinc finger protein 275 ZNF277 NM_021994 1307 ZnF-C2H2 zinc finger protein (C2H2 type) 277 ZNF278 NM_014323 1308 ZnF- zinc finger protein 278 BTB/POZ ZNF281 NM_012482 1309 ZnF-C2H2 zinc finger protein 281 ZNF282 D30612 1310 ZnF-C2H2 zinc finger protein 282 ZNF286 NM_020652 1311 ZnF-C2H2 zinc finger protein 286 ZNF287 NM_020653 1312 ZnF-C2H2 zinc finger protein 287 ZNF288 NM_015642 1313 ZnF- zinc finger protein 288 BTB/POZ ZNF29 X52357 1314 ZnF-C2H2 zinc finger protein 29 (KOX 26) ZNF294 AB018257 1315 ZnF-Other zinc finger protein 294 ZNF295 NM_020727 1316 ZnF- zinc finger protein 295 BTB/POZ ZNF297 NM_005453 1317 ZnF- zinc finger protein 297 BTB/POZ ZNF297B NM_014007 1318 ZnF- zinc finger protein 297B BTB/POZ ZNF3 NM_017715 1319 ZnF-C2H2 zinc finger protein 3 (A8-51) ZNF30 X52359 1320 ZnF-C2H2 zinc finger protein 30 (KOX 28) ZNF300 NT_006859:367 NM_052860 1321 ZnF-C2H2 zinc finger protein 300 ZNF302 NT_011196:498 NM_018443 1322 ZnF-C2H2 zinc finger protein 302 ZNF304 NM_020657 1323 ZnF-C2H2 zinc finger protein 304 ZNF305 NM_014724 1324 ZnF-C2H2 zinc finger protein 305 ZNF306 NM_024493 1325 ZnF-C2H2 zinc finger protein 306 ZNF31 NM_145238 1326 ZnF-C2H2 zinc finger protein 31 (KOX 29) ZNF313 NM_018683 1327 ZnF-Other zinc finger protein 313 ZNF317 NT_011176:75 NM_020933 1328 ZnF-C2H2 zinc finger protein 317 ZNF319 AB037809 1329 ZnF-C2H2 zinc finger protein 319 ZNF32 NM_006973 1330 ZnF-C2H2 zinc finger protein 32 (KOX 30) ZNF322A NT_007592:1565 NM_024639 1331 ZnF-PHD zinc finger protein 322A ZNF323 NT_007592:1771 NM_030899 1332 ZnF-C2H2 zinc finger protein 323 ZNF325 NM_016265 1333 ZnF-C2H2 zinc finger protein 325 ZNF333 NT_025155:3 NM_032433 1334 ZnF-C2H2 zinc finger protein 333 ZNF334 NM_018102 1335 ZnF-C2H2 zinc finger protein 334 ZNF335 NT_011362:859 NM_022095 1336 ZnF-C2H2 zinc finger protein 335 ZNF336 NT_011387:1856 NM_022482 1337 ZnF-C2H2 zinc finger protein 336 ZNF337 AL049942 1338 ZnF-C2H2 zinc finger protein 337 ZNF339 NT_011387:1400 NM_021220 1339 ZnF-C2H2 zinc finger protein 339 ZNF33A X68687 1340 ZnF-C2H2 zinc finger protein 33a (KOX 31) ZNF341 NT_028392:330 NM_032819 1341 ZnF-C2H2 zinc finger protein 341 ZNF342 NT_011109:256 NM_145288 1342 ZnF-C2H2 zinc finger protein 342 ZNF347 NT_011109:1491 NM_032584 1343 ZnF-C2H2 zinc finger protein 347 ZNF35 NM_003420 1344 ZnF-C2H2 zinc finger protein 35 (clone HF.10) ZNF350 NT_011109:1276 NM_021632 1345 ZnF-C2H2 zinc finger protein 350 ZNF354A NM_005649 1346 ZnF-C2H2 zinc finger protein 354A ZNF358 NM_018083 1347 ZnF-C2H2 zinc finger protein 358 ZNF36 U09848 1348 ZnF-C2H2 zinc finger protein 36 (KOX 18) ZNF361 NM_018555 1349 ZnF-C2H2 zinc finger protein 361 ZNF364 AL079314 1350 ZnF-Other zinc finger protein 364 ZNF366 NT_006713:99 NM_152625 1351 ZnF-C2H2 zinc finger protein 366 ZNF37A X69115 1352 ZnF-C2H2 zinc finger protein 37a (KOX 21) ZNF37A NT_033896:447 AJ492195 1353 ZnF-C2H2 zinc finger protein 37a (KOX21) ZNF38 NM_032924 1354 ZnF-C2H2 zinc finger protein 38 ZNF382 NT_011192:90 NM_032825 1355 ZnF-C2H2 zinc finger protein ZNF382 ZNF384 NT_009731:144 NM_133476 1356 ZnF-C2H2 zinc finger protein 384 ZNF394 NT_007933:1972 NM_032164 1357 ZnF-C2H2 zinc finger protein 394 ZNF396 NT_010934:143 NM_145756 1358 ZnF-C2H2 zinc finger protein 396 ZNF397 NT_010934:119 NM_032347 1359 ZnF-C2H2 zinc finger protein 397 ZNF398 NT_007914:756 NM_020781 1360 ZnF-C2H2 zinc finger protein 398 ZNF406 NT_007994:1 AB040918 1361 ZnF-C2H2 zinc finger protein 406 ZNF407 NT_025004:1 NM_017757 1362 ZnF-C2H2 zinc finger protein 407 ZNF408 NM_024741 1363 ZnF-C2H2 zinc finger protein 408 ZNF409 NT_025892:468 AB028979 1364 ZnF-C2H2 zinc finger protein 409 ZNF41 M92443 1365 ZnF-C2H2 zinc finger protein 41 ZNF42 NM_003422 1366 ZnF-C2H2 zinc finger protein 42 (myeloid-specific retinoic acid-responsive) ZNF426 NT_011176:123 NM_024106 1367 ZnF-C2H2 zinc finger protein 426 ZNF43 NM_003423 1368 ZnF-C2H2 zinc finger protein 43 (HTF6) ZNF431 NT_035560:82 NM_133473 1369 ZnF-C2H2 zinc finger protein 431 ZNF433 NT_011176:487 NM_152602 1370 ZnF-C2H2 zinc finger protein 433 ZNF434 NT_010552:596 NM_017810 1371 ZnF-C2H2 zinc finger protein 434 ZNF435 NT_007592:1726 NM_025231 1372 ZnF-C2H2 zinc finger protein 435 ZNF436 NT_032979:37 NM_030634 1373 ZnF-C2H2 zinc finger protein 436 ZNF44 X16281 1374 ZnF-C2H2 zinc finger protein 44 (KOX 7) ZNF440 NT_011176:446 NM_152357 1375 ZnF-AN1 zinc finger protein 440 ZNF443 NM_005815 1376 ZnF-C2H2 zinc finger protein 443 ZNF445 NT_034534:46 NM_181489 1377 ZnF-C2H2 zinc finger protein 445 ZNF45 NM_003425 1378 ZnF-C2H2 zinc finger protein 45 (a Kruppel-associated box (KRAB) domain polypeptide) ZNF454 NT_006802:20 NM_182594 1379 ZnF-C2H2 zinc finger protein 454 ZNF46 NM_006977 1380 ZnF- zinc finger protein 46 BTB/POZ (KUP) ZNF481 NT_017568:1387 NM_020924 1381 ZnF- zinc finger protein 481 BTB/POZ ZNF486 NT_035560:14 BC008936 1382 ZnF-C2H2 zinc finger protein 486 ZNF490 NT_011176:576 NM_020714 1383 ZnF-C2H2 zinc finger protein 490 ZNF491 NT_011176:438 NM_152356 1384 ZnF-C2H2 zinc finger protein 491 ZNF493 NT_035560:126b NM_175910 1385 ZnF-C2H2 zinc finger protein 493 ZNF494 NT_011104:214 NM_152677 1386 ZnF-C2H2 zinc finger protein 494 ZNF495 NT_011104:32a NM_024303 1387 ZnF-C2H2 zinc finger protein 495 ZNF496 NT_031730:64 NM_032752 1388 ZnF-C2H2 zinc finger protein 496 ZNF497 NT_011104:359 NM_198458 1389 ZnF-C2H2 zinc finger protein 497 ZNF498 NT_007933:1998 NM_145115 1390 ZnF-C2H2 zinc finger protein 498 ZNF502 NT_034534:1 NM_033210 1391 ZnF-C2H2 zinc finger protein 502 ZNF503 NT_033890:224 NM_032772 1392 ZnF-C2H2 zinc finger protein 503 ZNF509 NT_006051:22 NM_145291 1393 ZnF- zinc finger protein 509 BTB/POZ ZNF513 NT_005204:559 NM_144631 1394 ZnF-C2H2 zinc finger protein 513 ZNF514 NT_022300:33 NM_032788 1395 ZnF-C2H2 zinc finger protein 514 ZNF519 NT_010859:601 NM_145287 1396 ZnF-C2H2 zinc finger protein 519 ZNF528 NT_011109:1343 NM_032423 1397 ZnF-C2H2 zinc finger protein 528 ZNF6 NM_021998 1398 ZnF-C2H2 zinc finger protein 6 (CMPX1) ZNF7 NM_003416 1399 ZnF-C2H2 zinc finger protein 7 (KOX 4, clone HF.16) ZNF71 NT_011104:94 NM_021216 1400 ZnF-C2H2 zinc finger protein 71 (Cos26) ZNF73 NM_012480 1401 ZnF-C2H2 zinc finger protein 73 (Cos12) ZNF74 NM_003426 1402 ZnF-C2H2 zinc finger protein 74 (Cos52) ZNF75 NT_011786:383 NM_007131 1403 ZnF-C2H2 zinc finger protein 75 (D8C6) ZNF75A NM_153028 1404 ZnF-C2H2 zinc finger protein 75a ZNF76 NM_003427 1405 ZnF-C2H2 zinc finger protein 76 (expressed in testis) ZNF77 NT_011255:4 NM_021217 1406 ZnF-C2H2 zinc finger protein 77 (pT1) ZNF79 NM_007135 1407 ZnF-C2H2 zinc finger protein 79 (pT7) ZNF8 M29581 1408 ZnF-C2H2 zinc-finger protein 8 (clone HF.18) ZNF80 NM_007136 1409 ZnF-C2H2 zinc finger protein 80 (pT17) ZNF81 X68011 1410 ZnF-C2H2 zinc finger protein 81 (HFZ20) ZNF83 NM_018300 1411 ZnF-C2H2 zinc finger protein 83 (HPF1) ZNF84 NM_003428 1412 ZnF-C2H2 zinc finger protein 84 (HPF2) ZNF85 NM_003429 1413 ZnF-C2H2 zinc finger protein 85 (HPF4, HTF1) ZNF9 NM_003418 1414 ZnF-Other zinc finger protein 9 (a cellular retroviral nucleic acid binding protein) ZNF90 M61870 1415 ZnF-C2H2 zinc finger protein 90 (HTF9) ZNF91 NM_003430 1416 ZnF-C2H2 zinc finger protein 91 (HPF7, HTF10) ZNF92 M61872 1417 ZnF-C2H2 zinc finger protein 92 (HTF12) ZNF93 M61873 1418 ZnF-C2H2 zinc finger protein 93 (HTF34) ZNF-kaiso NM_006777 1419 ZnF- Kaiso BTB/POZ ZNFN1A1 NM_006060 1420 ZnF-C2H2 zinc finger protein, subfamily 1A, 1 (Ikaros) ZNFN1A2 NM_016260 1421 ZnF-C2H2 zinc finger protein, subfamily 1A, 2 (Helios) ZNFN1A3 NM_012481 1422 ZnF-C2H2 zinc finger protein, subfamily 1A, 3 (Aiolos) ZNFN1A4 NT_009458:35 NM_022465 1423 ZnF-MYND zinc finger protein, subfamily 1A, 4 (Eos) ZNF- NM_014415 1424 ZnF- zinc finger protein U69274 BTB/POZ ZNRF1 NT_035368:168 NM_032268 1425 ZnF-Other zinc and ring finger protein 1 ZXDA L14787 1426 ZnF-C2H2 zinc finger, X-linked, duplicated A ZXDB L14788 1427 ZnF-C2H2 zinc finger, X-linked, duplicated B ZYX NT_007914:428 NM_003461 1428 Co-activator zyxin

SOX2 (SEQ ID NO: 1501; NM_003106)    1 ggatggttgt ctattaactt gttcaaaaaa gtatcaggag ttgtcaaggc agagaagaga   61 gtgtttgcaa aagggggaaa gtagtttgct gcctctttaa gactaggact gagagaaaga  121 agaggagaga gaaagaaagg gagagaagtt tgagccccag gcttaagcct ttccaaaaaa  181 taataataac aatcatcggc ggcggcagga tcggccagag gaggagggaa gcgctttttt  241 tgatcctgat tccagtttgc ctctctcttt ttttccccca aattattctt cgcctgattt  301 tcctcgcgga gccctgcgct cccgacaccc ccgcccgcct cccctcctcc tctccccccg  361 cccgcgggcc ccccaaagtc ccggccgggc cgagggtcgg cggccgccgg cgggccgggc  421 ccgcgcacag cgcccgcatg tacaacatga tggagacgga gctgaagccg ccgggcccgc  481 agcaaacttc ggggggcggc ggcggcaact ccaccgcggc ggcggccggc ggcaaccaga  541 aaaacagccc ggaccgcgtc aagcggccca tgaatgcctt catggtgtgg tcccgcgggc  601 agcggcgcaa gatggcccag gagaacccca agatgcacaa ctcggagatc agcaagcgcc  661 tgggcgccga gtggaaactt ttgtcggaga cggagaagcg gccgttcatc gacgaggcta  721 agcggctgcg agcgctgcac atgaaggagc acccggatta taaataccgg ccccggcgga  781 aaaccaagac gctcatgaag aaggataagt acacgctgcc cggcgggctg ctggcccccg  841 gcggcaatag catggcgagc ggggtcgggg tgggcgccgg cctgggcgcg ggcgtgaacc  901 agcgcatgga cagttacgcg cacatgaacg gctggagcaa cggcagctac agcatgatgc  961 aggaccagct gggctacccg cagcacccgg gcctcaatgc gcacggcgca gcgcagatgc 1021 agcccatgca ccgctacgac gtgagcgccc tgcagtacaa ctccatgacc agctcgcaga 1081 cctacatgaa cggctcgccc acctacagca tgtcctactc gcagcagggc acccctggca 1141 tggctcttgg ctccatgggt tcggtggtca agtccgaggc cagctccagc ccccctgtgg 1201 ttacctcttc ctcccactcc agggcgccct gccaggccgg ggacctccgg gacatgatca 1261 gcatgtatct ccccggcgcc gaggtgccgg aacccgccgc ccccagcaga cttcacatgt 1321 cccagcacta ccagagcggc ccggtgcccg gcacggccat taacggcaca ctgcccctct 1381 cacacatgtg agggccggac agcgaactgg aggggggaga aattttcaaa gaaaaacgag 1441 ggaaatggga ggggtgcaaa agaggagagt aagaaacagc atggagaaaa cccggtacgc 1501 tcaaaaagaa aaaggaaaaa aaaaaatccc atcacccaca gcaaatgaca gctgcaaaag 1561 agaacaccaa tcccatccac actcacgcaa aaaccgcgat gccgacaaga aaacttttat 1621 gagagagatc ctggacttct ttttggggga ctatttttgt acagagaaaa cctggggagg 1681 gtggggaggg cgggggaatg gaccttgtat agatctggag gaaagaaagc tacgaaaaac 1741 tttttaaaag ttctagtggt acggtaggag ctttgcagga agtttgcaaa agtctttacc 1801 aataatattt agagctagtc tccaagcgac gaaaaaaatg ttttaatatt tgcaagcaac 1861 ttttgtacag tatttatcga gataaacatg gcaatcaaaa tgtccattgt ttataagctg 1921 agaatttgcc aatatttttc aaggagaggc ttcttgctga attttgattc tgcagctgaa 1981 atttaggaca gttgcaaacg tgaaaagaag aaaattattc aaatttggac attttaattg 2041 tttaaaaatt gtacaaaagg aaaaaattag aataagtact ggcgaaccat ctctgtggtc 2101 ttgtttaaaa agggcaaaag ttttagactg tactaaattt tataacttac tgttaaaagc 2161 aaaaatggcc atgcaggttg acaccgttgg taatttataa tagcttttgt tcgatcccaa 2221 ctttccattt tgttcagata aaaaaaacca tgaaattact gtgtttgaaa tattttctta 2281 tggtttgtaa tatttctgta aatttattgt gatattttaa ggttttcccc cctttatttt 2341 ccgtagttgt attttaaaag attcggctct gtattatttg aatcagtctg ccgagaatcc 2401 atgtatatat ttgaactaat atcatcctta taacaggtac attttcaact taagttttta 2461 ctccattatg cacagtttga gataaataaa tttttgaaat atggacactg aaaaaaaaaa FoxP3

The FOXP3 (forkhead box P3) gene encodes for a protein involved in immune system responses. A member of the FOX protein family, FOXP3 is a transcription factor that plays a role in the development and function of regulatory T cells. The induction or administration of Foxp3 positive T cells in animal studies indicate marked reductions in (autoimmune) disease severity in models of diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis and renal disease.

The FoxP3 protein can be expressed in a cell using the synthetic, modified RNAs described herein.

Targeting Moiety

As used herein, the term “targeting moiety” refers to an agent that directs a composition to a particular tissue, cell type, receptor, or other area of interest. As per this definition, a targeting moiety can be attached directly to a synthetic, modified RNA or indirectly to a composition used for delivering a synthetic, modified RNA (e.g., a liposome, polymer etc) to direct expression in a particular cell etc. A targeting moiety can also be encoded or expressed by a synthetic, modified-NA as described herein, such that a cell expresses a targeting moiety on it surface, permitting a cell to be targeted to a desired tissue, organ etc. For the avoidance of confusion, targeting moieties expressed on a cell surface are referred to herein as “homing moieties.”

Non-limiting examples of a targeting moiety or homing moiety include, but are not limited to, an oligonucleotide, an antigen, an antibody or functional fragment thereof, a ligand, a cell-surface receptor, a membrane-bound molecule, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative, a hormone, e.g., estradiol or histamine, a hormone-mimic, e.g., morphine, or hormone-receptor, or other compound having binding specificity for a target. In the methods of the present invention, a targeting moiety promotes transport or preferential localization of a synthetic, modified RNA to a target cell, while a homing moiety permits the targeting of a cell modified using the synthetic, modified RNAs described herein to a particular tissue in vivo. It is contemplated herein that the homing moiety can be also encoded in a cell by a synthetic, modified RNA as described herein.

A synthetic, modified RNA or composition thereof can be targeted by means of a targeting moiety, including, e.g., an antibody or targeted liposome technology. In some embodiments, a synthetic, modified RNA or composition thereof is targeted to a specific tissue by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of antibodies can be used for production of recombinant, bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules. The addition of an antibody to a synthetic, modified RNA composition permits the agent attached to accumulate additively at the desired target site. Antibody-based or non-antibody-based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site. Preferably, a natural binding agent for an unregulated or disease associated antigen is used for this purpose.

Table 2 and Table 3 provide non-limiting examples of CD (“cluster of differentiation”) molecules and other cell-surface/membrane bound molecules and receptors, such as transmembrane tyrosine kinase receptors, ABC transporters, and integrins, that can be expressed using the synthetic, modified RNA compositions and methods described herein for targeting and homing to cells of interest, or for changing the phenotype of a cell.

TABLE 2 List of CD Molecules Molecule (CD Number) NCBI Name NCBI Other Names CD10 MME CALLA; CD10; NEP CD100 SEMA4D CD100; M-sema G; M-sema-G; SEMAJ; coll-4 CD101 IGSF2 CD101; V7 CD102 ICAM2 CD102 CD103 ITGAE CD103; HUMINAE CD104 ITGB4 CD105 ENG CD105; END; HHT1; ORW; ORW1 CD106 VCAM1 INCAM-100 CD107a LAMP1 CD107a; LAMPA; LGP120 CD107b LAMP2 CD107b; LAMPB CD107b LAMP2 CD107b; LAMPB CD108 SEMA7A CD108; CDw108; H-SEMA-K1; H-Sema K1; H-Sema-L; SEMAK1; SEMAL CD109 CD109 DKFZp762L1111; FLJ38569 CD110 MPL C-MPL; CD110; MPLV; TPOR CD111 PVRL1 CD111; CLPED1; ED4; HIgR; HVEC; PRR; PRR1; PVRR; PVRR1; SK-12 CD112 PVRL2 CD112; HVEB; PRR2; PVRR2 CD113 PVRL3 PVTL3; PPR3; PRR3; PVRR3; nectin-3; DKFZP566B0846 CD114 CSF3R CD114; GCSFR CD115 CSF1R C-FMS; CD115; CSFR; FIM2; FMS CD116 CSF2RA CD116; CDw116; CSF2R; CSF2RAX; CSF2RAY; CSF2RX; CSF2RY; GM-CSF-R- alpha; GMCSFR; GMR; MGC3848; MGC4838 CD117 KIT CD117; PBT; SCFR CD118 LIFR LIFR; SWS; SJS2; STWS CD119 IFNGR1 CD119; IFNGR CD11a ITGAL CD11A; LFA-1; LFA1A CD11a ITGAL CD11A; LFA-1; LFA1A CD11a ITGAL CD11A; LFA-1; LFA1A CD11b ITGAM CD11B; CR3A; MAC-1; MAC1A; MO1A CD11c ITGAX CD11C CD11d ITGAD ADB2; CD11D CD120a TNFRSF1A CD120a; FPF; MGC19588; TBP1; TNF-R; TNF-R-I; TNF-R55; TNFAR; TNFR1; TNFR55; TNFR60; p55; p55-R; p60 CD120b TNFRSF1B CD 120b ; TBPII; TNF-R-II; TNF-R75; TNFBR; TNFR2; TNFR80; p75; p75TNFR CD121a IL1R1 CD121A; D2S1473; IL-1R-alpha; IL1R; IL1RA; P80 CD121b IL1R2 IL1RB; MGC47725 CD122 IL2RB P70-75 CD123 IL3RA CD123; IL3R; IL3RAY; IL3RX; IL3RY; MGC34174; hIL-3Ra CD124 IL4R CD124; IL4RA CD125 IL5RA CDw125; HSIL5R3; IL5R; MGC26560 CD126 IL6R CD126; IL-6R-1; IL-6R-alpha; IL6RA CD127 IL7R CD127; CDW127; IL-7R-alpha CD128a see CD181 see CD181 CD128b see CD182 see CD182 CD129 IL9R CD13 ANPEP CD13; LAP1; PEPN; gp150 CD130 IL6ST CD130; CDw130; GP130; GP130-RAPS; IL6R-beta CD131 CSF2RB CD131; CDw131; IL3RB; IL5RB CD132 IL2RG CD132; IMD4; SCIDX; SCIDX1 CD133 PROM1 AC133; CD133; PROML1 CD134 TNFRSF4 ACT35; CD134; OX40; TXGP1L CD135 FLT3 CD135; FLK2; STK1 CD136 MST1R CDw136; RON CD137 TNFRSF9 4-1BB; CD137; CDw137; ILA; MGC2172 CD138 SDC1 CD138; SDC; SYND1 CD139 CD139 CD14 CD14 CD14 CD14 CD140a PDGFRA CD140A; PDGFR2 CD140b PDGFRB CD140B; JTK12; PDGF-R-beta; PDGFR; PDGFR1 CD141 THBD CD141; THRM; TM CD142 F3 CD142; TF; TFA CD143 ACE ACE1; CD143; DCP; DCP1; MGC26566 CD144 CDH5 7B4 CD146 MCAM CD146; MUC18 CD147 BSG 5F7; CD147; EMMPRIN; M6; OK; TCSF CD148 PTPRJ CD148; DEP1; HPTPeta; R-PTP-ETA; SCC1 CD149 see CD47R see CD47R CD15 FUT4 CD15; ELFT; FCT3A; FUC-TIV CD15 FUT4 CD15; ELFT; FCT3A; FUC-TIV CD15 FUT4 CD15; ELFT; FCT3A; FUC-TIV CD150 SLAMF1 CD150; CDw150; SLAM CD151 CD151 GP27; PETA-3; SFA1 CD152 CTLA4 CD152 CD153 TNFSF8 CD153; CD30L; CD30LG CD154 CD40LG CD154; CD40L; CD40LG; HIGM1; IGM; IMD3; T-BAM; TRAP; gp39; hCD40L CD155 PVR CD155; HVED; NECL5; PVS; TAGE4 CD156a ADAM8 CD156; MS2 CD156b ADAM17 CD156b; TACE; cSVP CD156C ADAM10 kuz; MADM; CD156c; HsT18717 CD157 BST1 CD157 CD158A KIR2DL1 47.11; CD158A; CL-42; NKAT1; p58.1 CD158B1 KIR2DL2 CD158B1; CL-43; NKAT6; p58.2 CD158B2 KIR2DL3 CD158B2; CD158b; CL-6; KIR-023GB; NKAT2; NKAT2A; NKAT2B; p58 CD158C KIR3DP1; LOC392419 KIR2DS6; KIRX CD158D KIR2DL4 103AS; 15.212; CD158D; KIR103; KIR103AS CD158E1 KIR3DL1 AMB11; CD158E1; CD158E1/2; CD158E2; CL-11; CL-2; KIR; KIR3DS1; NKAT10; NKAT3; NKB1; NKB1B CD158E2 KIR3DS1 AMB11; CD158E1; CD158E1/2; CD158E2; CL-11; CL-2; KIR; KIR3DS1; NKAT10; NKAT3; NKB1; NKB1B CD158F KIR2DL5 CD158F; KIR2DL5; KIR2DL5.1; KIR2DL5.3 CD158G KIR2DS5 CD158G; NKAT9 CD158H KIR2DS1 CD158H; EB6ActI; EB6ActII; p50.1 CD158I KIR2DS4 CD158I; KIR1D; KKA3; NKAT8; PAX; c1-39 CD158J KIR2DS2 183ACTI; CD158J; CL-49; NKAT5; p50.2 CD158K KIR3DL2 CD158K; CL-5; NKAT4; NKAT4A; NKAT4B CD159a KLRC1 CD159A; MGC13374; MGC59791; NKG2; NKG2A CD159c KLRC2 CD160 CD160 BY55; NK1; NK28 CD161 KLRB1 CD161; NKR; NKR-P1; NKR-P1A; NKRP1A; hNKR-P1A CD162 SELPLG CD162; PSGL-1; PSGL1 CD163 CD163 M130; MM130 CD164 CD164 MGC-24; MUC-24; endolyn CD165 CD165 CD166 ALCAM CD166; MEMD CD167a DDR1 CAK; CD167; DDR; EDDR1; MCK10; NEP; NTRK4; PTK3; PTK3A; RTK6; TRKE CD167b DDR2 TKT; MIG20a; NTRKR3; TYRO10 CD168 HMMR RHAMM CD169 SN CD169; FLJ00051; FLJ00055; FLJ00073; FLJ32150; SIGLEC-1; dJ1009E24.1 CD16a FCGR3A CD16; FCG3; FCGR3; IGFR3 CD16b FCGR3B CD16; FCG3; FCGR3 CD17 carbohydrate carbohydrate CD170 SIGLEC5 CD33L2; OB-BP2; OBBP2; SIGLEC-5 CD171 L1CAM CAML1; CD171; HSAS; HSAS1; MASA; MIC5; N-CAML1; S10; SPG1 CD172a PTPNS1 BIT; MIR; MYD-1; P84; SHPS-1; SHPS1; SIRP; SIRP-ALPHA-1; SIRPalpha; SIRPalpha2 CD172b SIRPB1 SIRP-BETA-1 CD172g SIRPB2 SIRP-B2; bA77C3.1 CD173 carbohydrate carbohydrate CD174 FUT3 LE; Les CD175 carbohydrate carbohydrate CD175s carbohydrate carbohydrate CD176 carbohydrate carbohydrate CD177 CD177 CD177; HNA2A; NB1 CD178 FASLG FASL; CD178; CD95L; TNFSF6; APT1LG1 CD179a VPREB1 IGI; IGVPB; VPREB CD179b IGLL1 14.1; CD179b; IGL1; IGL5; IGLL; IGO; IGVPB; VPREB2 CD18 ITGB2 CD18; LAD; LCAMB; LFA-1; MF17; MF17 CD180 CD180 LY64; Ly78; RP105; MGC126233; MGC126234 CD181 IL8RA C-C CKR-1; C-C-CKR-1; CD128; CDw128a; CMKAR1; CXCR1; IL8R1; IL8RBA CD182 IL8RB CDw128b; CMKAR2; CXCR2; IL8R2; IL8RA CD183 CXCR3 CD183; CKR-L2; CMKAR3; GPR9; IP10; IP10-R; Mig-R; MigR CD184 CXCR4 D25201E; HM89; HSY3RR; LAP3; LESTR; NPY3R; NPYR; NPYY3R; WHIM CD185 BLR1 BLR1; CXCR5; MDR15 CD186 CXCR6 CXCR6; BONZO; STRL33; TYMSTR CD187 CD188 CD189 CD19 CD19 B4; MGC12802 CD190 CD191 CCR1 CKR-1; CMKBR1; HM145; MIP1aR; SCYAR1 CD192 CCR2 CC-CKR-2; CCR2A; CCR2B; CKR2; CKR2A; CKR2B; CMKBR2; MCP-1-R CD193 CCR3 CC-CKR-3; CKR3; CMKBR3 CD194 CCR4 CC-CKR-4; CKR4; CMKBR4; ChemR13; HGCN CD195 CCR5 CC-CKR-5; CCCKR5 ; CD195; CKR-5; CKR5 ; CMKBR5 CD196 CCR6 CCR6; BN-1; CKR6; DCR2; CKRL3; DRY-6; GPR29; CKR-L3; CMKBR6; GPRCY4; STRL22; GPR-CY4 CD197 CCR7 BLR2; CDw197; CMKBR7; EBIl CD1a CD1A CD1 CD1b CD1B CD1 CD1c CD1C CD1 CD1d CD1D CD1d CD1D CD1e CD1E HSCDIEL CD2 CD2 SRBC; T11 CD2 CD2 SRBC; T11 CD20 MS4A1 B1; Bp35; CD20; LEU-16; MGC3969; MS4A2; S7 CD200 CD200 MOX1; MOX2; MRC; OX-2 CD201 PROCR CCCA; CCD41; EPCR; MGC23024; bA4204.2 CD202b ILK CD202B; TIE-2; TIE2; VMCM; VMCM1 CD203c ENPP3 B10; CD203c; NPP3; PD-IBETA; PDNP3 CD204 MSR1 SCARA1; SR-A; phSR1; phSR2 CD205 LY75 CLEC13B; DEC-205; GP200-MR6 CD206 MRC1 CLEC13D CD207 CD207 LANGERIN CD208 LAMP3 DC-LAMP; DCLAMP; LAMP; T5C403 CD209 CD209 CDSIGN; DC-SIGN; DC-SIGN1 CD21 CR2 C3DR; CD21 CD211 CD212 IL12RB1 IL-12R-BETA1; IL12RB; MGC34454 CD213a1 IL13RA1 IL-13Ra; NR4 CD213a2 IL13RA2 IL-13R; IL13BP CD214 CD215 CD216 CD217 IL17R IL-17RA; IL17RA; MGC10262; hIL-17R CD218a IL18R1 11_,18R1; IL1RRP; IL-1Rrp CD218b IL18RAP IL18RAP; ACPL CD219 CD22 CD22 SIGLEC-2 CD220 INSR CD221 IGF1R JTK13 CD222 IGF2R CD222; CIMPR; M6P-R; MPRI CD223 LAG3 CD223 CD224 GGT1 CD224; D22S672; D22S732; GGT; GTG CD225 IFITM1 Sep-27; CD225; IF117; LEU13 CD226 CD226 DNAM-1; DNAM1; PTA1; TLiSA1 CD227 MUC1 CD227; EMA; PEM; PUM CD228 MF12 MAP97; MGC4856; MTF1 CD229 LY9 CD229; SLAMF3; hly9; mLY9 CD23 FCER2 CD23; CD23A; FCE2; IGEBF CD230 PRNP ASCR; CJD; GSS; MGC26679; PRIP; PrP; PrP27-30; PrP33-35C; PrPc CD231 TSPAN7 A15; CCG-B7; CD231; DX51692E; MXS1; TALLA-1; TM4SF2b CD232 PLXNC1 PLXN-C1; VESPR CD233 SLC4A1 AE1; BND3; CD233; DI; EMPB3; EPB3; RTA1A; WD; WD1 CD234 DARC CCBP1; DARC; GPD CD235a GYPA GPA; MN; MNS CD235b GYPB GPB; MNS; SS CD236 GYPC GE; GPC CD237 CD238 KEL CD239 LU AU; BCAM; MSK19 CD24 CD24 CD24A CD240CE RHCE RH; RH30A; RHC; RHE; RHIXB; RHPI; Rh4; RhVI; RhVIII CD240D RHD CD240D; DIIIc; RH; RH30; RHCED; RHDVA(TT); RHPII; RHXIII; Rh30a; Rh4; RhII; RhK562-II; RhPI CD241 RHAG RH2; RH50A CD242 ICAM4 LW CD243 ABCB1 ABC20; CD243; CLCS; GP170; MDR1; P-gp; PGY1 CD244 CD244 2B4; NAIL; NKR2B4; Nmrk; SLAMF4 CD245 CD245 CD246 ALK CD247 CD247 CD3-ZETA; CD3H; CD3Q; TCRZ CD248 CD248 CD164L1 CD249 ENPEP APA; gp160; EAP CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD250 CD251 CD252 TNFSF4 TNFSF4; GP34; OX4OL; TXGP1; CD134L; OX-40L; OX4OL CD253 TNFSF10 TNFSF10; TL2; APO2L; TRAIL; Apo-2L CD254 TNFSF11 ODF; OPGL; sOdf; CD254; OPTB2; RANKL; TRANCE; 1iRANKL2 CD255 CD256 TNFSF13 APRIL; TALL2; TRDL -1; UNQ383/PRO715 CD257 TNFSF13B BAFF; BLYS; TALL-1; TALL1; THANK; TNFSF20; ZTNF4; delta BAFF CD258 TNFSF14 TNFSF14; LTg; TR2; HVEML; LIGHT CD259 CD26 DPP4 ADABP; ADCP2; CD26; DPPIV; TP103 CD260 CD261 TNFRSF10A APO2; DR4; MGC9365; TRAILR-1; TRAILR1 CD262 TNFRSF10B DR5; KILLER; KILLER/DR5; TRAIL-R2; TRAILR2; TRICK2; TRICK2A; TRICK2B; TRICKB; ZTNFR9 CD263 TNFRSF10C DCR1; LIT; TRAILR3; TRID CD264 TNFRSF10D DCR2; TRAILR4; TRUNDD CD265 TNFRSF11A EOF; FEO; ODFR; OFE; PDB2; RANK; TRANCER CD266 TNFRSF12A TNFRSF12A; FN14 ; TWEAKR CD267 TNFRSF13B CVID; TACI; CD267; FLJ39942; MGC39952; MGC133214; TNFRSF14B CD268 TNFRSF13C BAFFR; CD268; BAFF-R; MGC138235 CD269 TNFRSF17 BCM; BCMA CD27 TNFRSF7 CD27; MGC20393; S152; T14; Tp55 CD270 CD271 NGFR NGFR; TNFRSF16; p75(NTR) CD272 BTLA BTLA1; FLJ16065 CD273 PDCD1LG2 PDCD1LG2; B7DC; Btdc; PDL2; PD-L2; PDCD1L2; bA574F11.2 CD274 CD274 B7-H; B7H1; PD-Ll; PDCD1L1; PDL1 CD275 ICOSLG B7-H2; B7H2; B7RP-1; B7RP1; GL50; ICOS-L; ICOSLG; KIAA0653; LICOS CD276 CD276 B7H3 CD277 BTN3A1 BTF5; BT3.1 CD278 ICOS AILIM; MGC39850 CD279 PDCD1 PD1; SLEB2; hPD-1 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD280 MRC2 MRC2; UPARAP; ENDO180; KIAA0709 CD281 TLR1 TLR1; TIL; rsc786; KIAA0012; DKFZp547I0610; DKFZp564I0682 CD282 TLR2 TIL4 CD283 TLR3 TLR3 CD284 TLR4 TOLL; hToll CD285 CD286 TLR6 CD286 CD287 CD288 TLR8 TLR8 CD289 TLR9 none CD29 ITGB1 CD29; FNRB; GPIIA; MDF2; MSK12; VLAB CD290 TLR10 TLR10 CD291 CD292 BMPR1A BMPR1A; ALK3; ACVRLK3 CD294 GPR44 CRTH2 CD295 LEPR LEPR; OBR CD296 ART1 ART2; RT6 CD297 ART4 DO; DOK1; CD297; ART4 CD298 ATP1B3 ATP1B3; ATPB-3; F1129027 CD299 CLEC4M DC-SIGN2; DC-SIGNR; DCSIGNR; HP10347; LSIGN; MGC47866 CD3 see CD3D, see CD3D, CD3E, CD3G CD3E, CD3G CD3 see CD3D, see CD3D, CD3E, CD3G CD3E, CD3G CD30 TNFRSF8 CD30; D1S166E; KI-1 CD300a CD300A CMRF-35-H9; CMRF35H; CMRF35H9; IRC1; IRC2; IRp60 CD300C CD300C CMRF-35A; CMRF35A; CMRF35A1; LIR CD301 CLEC10A HML; HML2; CLECSF13; CLECSF14 CD302 CD302 DCL-1; BIMLEC; KIAA0022 CD303 CLEC4C BDCA2; CLECSF11; DLEC; HECL; PR034150; CLECSF7 CD304 NRP1 NRP; VEGF165R CD305 LAIR1 LAIR-1 CD306 LAIR2 LAIR2 CD307 FCRL5 BXMAS1 CD308 CD309 KDR KDR; FLK1; VEGFR; VEGFR2 CD31 PECAM1 CD31 CD31 PECAM1 CD31 CD31 PECAM1 CD31 CD310 CD311 CD312 EMR2 CD313 CD314 KLRK1 KLRK1; KLR; NKG2D; NKG2-D; D12S2489E CD315 PTGFRN PTGFRN; FPRP; EWI-F; CD9P-1; SMAP-6; FLJ11001; KIAA1436 CD316 IGSF8 IGSF8; EWI2; PGRL; CD81P3 CD317 BST2 none CD318 CDCP1 CDCP1; F1122969; MGC31813 CD319 SLAMF7 19A; CRACC; CS1 CD320 CD320 8D6A; 8D6 CD321 F11R JAM; KAT; JAM1; JCAM; JAM-1; PAM-1 CD322 JAM2 C21orf43; VE-JAM; VEJAM CD323 CD324 CDH1 Arc-1; CDHE; ECAD; LCAM; UVO CD325 CDH2 CDHN; NCAD CD326 TACSTD1 CO17-1A; EGP; EGP40; Ep-CAM; GA733-2; KSA; M4S1; MIC18; MK-1; TROP1; hEGP-2 CD327 SIGLEC6 CD33L; CD33L1; OBBP1; SIGLEC-6 CD328 SIGLEC7 p75; QA79; AIRM1; CDw328; SIGLEC-7; p75/AIRM1 CD329 SIGLEC9 CDw329; OBBP-LIKE CD32a FCGR2A CD32; CDw32; FCG2; FCGR2; FCGR2A1; FcGR; IGFR2; MGC23887; MGC30032 CD32b FCGR2B CD32; FCG2; FCGR2; IGFR2 CD32c FCGR2C CD32; FcgammaRIIC CD33 CD33 SIGLEC-3; p67 CD33 CD33 SIGLEC-3; p67 CD330 CD331 FGFR1 FGFR1; H2; H3; H4; H5; CEK; FLG; FLT2; KAL2; BFGFR; C-FGR; N-SAM CD332 FGFR2 FGFR2; BEK; JWS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1; K-SAM CD333 FGFR3 FGFR3; ACH; CEK2; JTK4; HSFGFR3EX CD334 FGFR4 FGFR4; TKF; JTK2; MGC20292 CD335 NCR1 LY94; NK-p46; NKP46 CD336 NCR2 LY95; NK-p44; NKP44 CD337 NCR3 1C7; LY117; NKp30 CD338 ABCG2 MRX; MXR; ABCP; BCRP; BMDP; MXR1; ABC15; BCRP1; CDw338; EST157481; MGC102821 CD339 JAG1 JAG1; AGS; AHD; AWS; HJ1; JAGL1 CD34 CD34 CD34 CD34 CD340 ERBB2 NEU; NGL; HER2; TKR1; HER-2; c-erb B2; HER-2/neu CD344 FZD4 EVR1; FEVR; Fz-4; FzE4; GPCR; FZD4S; MGC34390 CD349 FZD9 FZD3 CD35 CR1 C3BR; CD35 CD350 FZD10 FzE7; FZ-10; hFz10 CD36 CD36 FAT; GP3B; GP4; GPIV; PASIV; SCARB3 CD37 CD37 GP52-40 CD38 CD38 T10 CD39 ENTPD1 ATPDase; CD39; NTPDase-1 CD3d CD3D CD3-DELTA; T3D CD3e CD3E CD3-EPSILON; T3E; TCRE CD3g CD3G CD3-GAMMA; T3G CD4 CD4 CD4 CD4 CD40 CD40 p50; Bp50; CDW40; MGC9013; TNFRSF5 CD41 ITGA2B CD41; CD41B; GP2B; GPIIb; GTA CD42a GP9 CD42a CD42b GP1BA BSS; CD42B; CD42b-alpha; GP1B; MGC34595 CD42c GP1BB CD42c CD42d GP5 CD42d CD43 SPN CD43; GPL115; LSN CD43 SPN CD43; GPL115; LSN CD43 SPN CD43; GPL115; LSN CD43 SPN CD43; GPL115; LSN CD44 CD44 CDW44; ECMR-III; IN; INLU; LHR; MC56; MDU2; MDU3; MGC10468; MIC4; MUTCH-I; Pgp1 CD44 CD44 CDW44; ECMR-III; IN; INLU; LHR; MC56; MDU2; MDU3; MGC10468; MIC4; MUTCH-I; Pgp1 CD44 CD44 CDW44; ECMR-III; IN; INLU; LHR; MC56; MDU2; MDU3; MGC10468; MIC4; MUTCH-I; Pgp1 CD45 PTPRC B220; CD45; GP180; LCA; LY5; T200 CD45RA PTPRC CD45RB PTPRC CD45RC PTPRC CD45RO PTPRC CD46 MCP CD46; MGC26544; MIC10; TLX; TRA2.10 CD47 CD47 IAP; MER6; OA3 CD48 CD48 BCM1; BLAST; BLAST1; MEM-102; SLAMF2; hCD48; mCD48 CD49a ITGA1 CD49a; VLA1 CD49b ITGA2 BR; CD49B; VLAA2 CD49c ITGA3 CD49C; GAP-B3; GAPB3; M5K18; VCA-2; VL3A; VLA3a CD49d ITGA4 CD49D CD49e ITGA5 CD49e; FNRA; VLA5A CD49f ITGA6 CD49f CD5 CD5 LEU1; T1 CD5 CD5 LEU1; T1 CD50 ICAM3 CD50; CDW50; ICAM-R CD51 ITGAV CD51; MSK8; VNRA CD52 CD52 CD52 CD53 CD53 MOX44 CD54 ICAM1 BB2; CD54 CD55 DAF CD55; CR; TC CD56 NCAM1 CD56; M5K39; NCAM CD57 CD57 HNK-1; LEU7; NK-1 CD58 CD58 LFA3 CD59 CD59 MGC2354; MIC11; MINI; MIN2; MIN3; M5K21; PROTECTIN CD6 CD6 TP120 CD6 CD6 TP120 CD60a carbohydrate carbohydrate CD60b carbohydrate carbohydrate CD60b carbohydrate carbohydrate CD60c carbohydrate carbohydrate CD61 ITGB3 CD61; GP3A; GPIIIa CD62E SELE CD62E; ELAM; ELAM1; ESEL; LECAM2 CD62L SELL CD62L; LAM-1; LAM1; LECAM1; LNHR; LSEL; LYAM1; Leu-8; Lyam-1; PLNHR; TQ1; hLHRc CD62P SELP CD62; CD62P; GMP140; GRMP; PADGEM; PSEL CD63 CD63 LAMP-3; ME491; MLA1; 0MA81H CD64a FCGR1A CD64; FCRI; IGFR1 CD65 carbohydrate carbohydrate CD65s carbohydrate carbohydrate CD66a CEACAM1 BGP; BGP1; BGPI; CD66; CD66A CD66b CEACAM8 CD66b; CD67; CGM6; NCA-95 CD66c CEACAM6 CD66c; CEAL; NCA CD66d CEACAM3 CD66D; CGM1 CD66e CEACAM5 CD66e; CEA CD66f PSG1 B1G1; CD66f; PBG1; PSBG1; PSGGA; SP1 CD67 see CD66f see CD66f CD68 CD68 SCARD1 CD69 CD69 none CD7 CD7 GP40; LEU-9; TP41; Tp40 CD7 CD7 GP40; LEU-9; TP41; Tp40 CD70 TNFSF7 CD27L; CD27LG; CD70 CD71 TFRC CD71; TFR; TRFR CD72 CD72 LYB2 CD73 NT5E CD73; E5NT; NT5; NTE; eN; eNT CD74 CD74 DHLAG; HLADG; Ia-GAMMA CD75 carbohydrate carbohydrate CD75s carbohydrate carbohydrate CD76 see CD75 and see CD75 and CD75s CD75s CD77 carbohydrate carbohydrate CD78 deleted deleted CD79a CD79A IGA; MB-1 CD79b CD79B B29; IGB CD80 CD80 CD28LG; CD28LG1; LAB7 CD81 CD81 S5.7; TAPA1 CD82 CD82 4F9; C33; CD82; GR15; IA4; R2; SAR2; ST6 CD83 CD83 BL11; HB15 CD84 CD84 LY9B; SLAMF5; hCD84; mCD84 CD85A LILRB3 CD85A; HL9; ILT5; LIR-3; LIR3 CD85B LILRB6 LILRB6 CD85C LILRB5 CD85C; LIR-8; LIR8 CD85D LILRB2 CD85D; ILT4; LIR-2; LIR2; MIR-10; MIR10 CD85E LILRA3 CD85E; HM31; HM43; ILT6; LIR-4; LIR4 CD85F LILRB7 CD85F; ILT11; LILRB7 CD85G LILRA4 ILT7; CD85g; MGC129597 CD85H LILRA2 CD85H; ILT1; LIR-7; LIR7 CD85I LILRA1 CD85I; LIR-6; LIR6 CD85J LILRB1 CD85; CD85J; ILT2; LIR-1; LIR1; MIR-7; MIR7 CD85K LILRB4 CD85K; HM18; ILT3; LIR-5; LIR5 CD85L LILRP1 ILT9; CD851; LILRA6P CD85M LILRP2 CD85m; ILT10; LILRA5 CD86 CD86 B7-2; B70; CD28LG2; LAB72; MGC34413 CD87 PLAUR CD87; UPAR; URKR CD88 C5R1 C5A; C5AR; CD88 CD89 FCAR CD89 CD8a CD8A CD8; Leu2; MAL; p32 CD8a CD8A CD8; Leu2; MAL; p32 CD8b CD8B1 CD8B; LYT3; Leu2; Ly3 CD9 CD9 BA2; DRAP-27; MIC3; MRP-1; P24 CD90 THY1 CD90 CD91 LRP1 A2MR; APOER; APR; CD91; LRP CD92 SLC44A1 CTL1; CDW92; CHTL1; RP11-287A8.1 CD93 CD93 C1QR1; C1qRP; CDw93; MXRA4; ClqR(P); dJ737E23.1 CD94 KLRD1 CD94 CD95 FAS APT1; CD95; FAS1; APO-1; FASTM; ALPS1A; TNFRSF6 CD96 CD96 MGC22596; TACTILE CD97 CD97 TM7LN1 CD98 SLC3A2 4F2; 4F2HC; 4T2HC; CD98; MDUl; NACAE CD99 CD99 MIC2; MIC2X; MIC2Y CD99R CD99 CDW12 CDw12 CDw12; p90-120 CDw145 CDw145 not listed CDw198 CCR8 CKR-L1; CKRL1; CMKBR8; CMKBRL2; CY6; GPR-CY6; IER1 CDw199 CCR9 GPR-9-6; GPR28 CDw210a IL10RA CDW210A; HIL-10R; IL-10R1; IL10R CDw210b IL10RB CDW210B; CRF2-4; CRFB4; D21S58; D21S66; IL-10R2 CDw293 BMPR1B BMPR1B; ALK6; ALK-6

TABLE 3 List of Membrane-Bound Receptors Membrane-bound Receptor Name mRNA ID 5-HT3 receptor subunit E splice DQ644022.1 variant HTR3Ea 5-HT3 serotonin receptor (long AJ003078.1 isoform) 5-HT3c1 serotonin receptor-like AY349352.1 protein AY349353.1 5-hydroxytryptamine (serotonin) BC101091.2 BC101090.2 receptor 3 family member D NM_001145143.1 NM_182537.2 AJ437318.1 AY159812.2 GI:110431739 5-hydroxytryptamine (serotonin) NM_130770.2 receptor 3, family member C BC131799.1 (HTR3C) AF459285.1 5-hydroxytryptamine (serotonin) NM_182589.2 receptor 3, family member E BC101183.2 (HTR3E) BC101185.2 BC101182.1 AY159813.2 EU165354.1 5-hydroxytryptamine (serotonin) BC004453.1 receptor 3A (HTR3A) BC002354.2 BT007204.1 GI:30583246 NM_001161772.2 NM_213621.3 NM_000869.5 AF498984.1 5-hydroxytryptamine (serotonin) NM_006028.3 receptor 3B (HTR3B) AK314268.1 AF169255.1 AF080582.1 AM293589.1 ABA-A receptor, alpha 1 subunit X14766.1 ABC protein AF146074.1 ABC transporter 7 protein AB005289.1 ABC transporter MOAT-B (MOAT-B) AF071202.1 ABC transporter MOAT-C (MOAT-C) AF104942.1 ABC transporter MOAT-D (MOAT-D) AF104943.1 ABC transporter umat (ABCB6 gene) AJ289233.2 ABCB5 mRNA for ATP-binding AB353947.1 cassette, sub-family B (MDR/TAP), member 5 ABCC4 protein AB208973.1 acetylcholine receptor (epsilon subunit) X66403.1 acetylcholine receptor delta subunit X55019.1 GI:297401 adrenoleukodystrophy related protein AJ000327.1 (ALDR) ALD gene Z21876.1 alpha 7 neuronal nicotinic acetylcholine U40583.1 receptor alpha-1 strychnine binding subunit of X52009.1 inhibitory glycine receptor mRNA alpha-2 strychnine binding subunit of X52008.1 inhibitory glycine receptor mRNA alpha-3 neuronal nicotinic acetylcholine M37981.1 receptor subunit amino butyric acid (GABA rho2) gene M86868.1 amino butyric acid (GABAA) receptor M82919.1 beta-3 subunit amma-aminobutyric acid (GABA) BC130344.1 receptor, rho 1 Anaplastic lymphoma receptor tyrosine NM_004304.4 kinase (ALK) anthracycline resistance associated X95715.1 protein ATP binding cassette transporter AF038950.1 ATP-binding cassette (sub-family C, AM774324.1 member 6) (ABCC6 gene) AM711638.1 ATP-binding cassette 7 iron transporter AF133659.1 (ABC7) ATP-binding cassette C5 AB209103.1 ATP-binding cassette half-transporter AF308472.1 (PRP) ATP-binding cassette protein (ABCB5) AY230001.1 AY196484.1 ATP-binding cassette protein ABCB9 AF216494.1 (ABCB9) ATP-binding cassette protein C11 AF367202.1 (ABCC11) AF411579.1 AY040219.1 NM_003742.2 ATP-binding cassette protein C12 AF395909.1 (ABCC12) AF411578.1 AF411577.1 AF395908.1 AY040220.1 ATP-binding cassette protein C13 AY063514.1 AF518320.1 ATP-binding cassette protein M-ABC1 AF047690.1 ATP-binding cassette subfamily B AY785909.1 AY851365.1 member 5 (ABCB5) ATP-binding cassette transporter C4 AY207008.1 AF541977.1 (ABCC4) ATP-binding cassette transporter MRP8 AF352582.1 ATP-binding cassette, sub-family B BC130424.1 (MDR/TAP), member 1 (ABCB1) NM_000927.4 ATP-binding cassette, sub-family B BC064930.1 (MDR/TAP), member 10 (ABCB10) NM_012089.2 NM_001198934.1 ATP-binding cassette, sub-family B BC042531.1 (MDR/TAP), member 4 (ABCB4) BC020618.2 NM_018849.2 NM_000443.3 NM_018850.2 ATP-binding cassette, sub-family B BC104894.1 (MDR/TAP), member 5 (ABCB5) BC104920.1 NM_001163941.1 NM_178559.5 ATP-binding cassette, sub-family B BC000559.2 (MDR/TAP), member 6 (ABCB6) NM_005689.2 ATP-binding cassette, sub-family B BC006323.2 (MDR/TAP), member 7 (ABCB7) BT009918.1 NM_004299.3 ATP-binding cassette, sub-family B BC151235.1 BC141836.1 (MDR/TAP), member 8 (ABCB8) BGI:146327013 NM_007188.3 AK222911.1 ATP-binding cassette, sub-family B BC017348.2 (MDR/TAP), member 9 (ABCB9) BC064384.1 NM_019624.3 NM_019625.3 NM_203444.2 ATP-binding cassette, sub-family C NM_019898.2 (CFTR/MRP), member 1 (ABCC1) NM_019899.2 NM_019862.2 NM_004996.3 NM_019900.2 AB209120.1 ATP-binding cassette, sub-family C NM_033450.2 GI:25914748 (CFTR/MRP), member 10 (ABCC10) ATP-binding cassette, sub-family C NM_145186.2 (CFTR/MRP), member 11 (ABCC11) NM_032583.3 NM_033151.3 ATP-binding cassette, sub-family C NM_033226.2 (CFTR/MRP), member 12 (ABCC12) ATP-binding cassette, sub-family C BC136419.1 GI:187953242 (CFTR/MRP), member 2 (ABCC2) NM_000392.3 ATP-binding cassette, sub-family C BC046126.1 (CFTR/MRP), member 3 (ABCC3) BC137347.1BC137348.1 BC104952.1 BC050370.1 NM_001144070.1 NM_003786.3 AB208954.1 ATP-binding cassette, sub-family C BC041560.1 (CFTR/MRP), member 4 (ABCC4) NM_001105515.1 NM_005845.3 ATP-binding cassette, sub-family C BC140771.1 (CFTR/MRP), member 5 (ABCC5) NM_005688.2 ATP-binding cassette, sub-family C BC131732.1 (CFTR/MRP), member 6 (ABCC6) NM_001171.5 ATP-binding cassette, sub-family C NM_000352.3 (CFTR/MRP), member 8 (ABCC8) ATP-binding cassette, sub-family C NM_020298.2 NM_020297.2 (CFTR/MRP), member 9 (ABCC9) NM_005691.2 ATP-binding cassette, sub-family D BC025358.1 (ALD), member 1 (ABCD1) BC015541.1 NM_000033.3 ATP-binding cassette, sub-family D BC104901.1 (ALD), member 2 (ABCD2) BC104903.1 NM_005164.3 AK314254.1 ATP-binding cassette, sub-family D BC009712.2 (ALD), member 3 (ABCD3) BC068509.1 BT006644.1 NM_001122674.1 NM_002858.3 ATP-binding cassette, sub-family D BC012815.2 (ALD), member 4 (ABCD4) BT007412.1 NM_005050.3 beta 4 nicotinic acetylcholine receptor U48861.1 subunit bile salt export pump (BSEP) AF136523.1 AF091582.1 B-lymphocyte CR2-receptor (for Y00649.1 complement factor C3d and Epstein- Barr virus) Butyrophilin-like 2 (MHC class II NM_019602.1 associated) (BTNL2) Cadherin 1, type 1, E-cadherin NM_004360.3 (epithelial) (CDH1) Cadherin 13, H-cadherin (heart) NM_001257.3 (CDH13) Cadherin 15, type 1, M-cadherin NM_004933.2 (myotubule) (CDH15) Cadherin 16, KSP-cadherin (CDH16) NM_001204746.1 NM_001204745.1 NM_001204744.1 NM_004062.3 Cadherin 17, LI cadherin (liver- NM_001144663.1 intestine) (CDH17) NM_004063.3 Cadherin 19, type 2 (CDH19) NM_021153.2 Cadherin 2, type 1, N-cadherin NM_001792.3 (neuronal) (CDH2) cadherin 20, type 2 (CDH20) NM_031891.2 Cadherin 3, type 1, P-cadherin (CDH3) NM_001793.4 Cadherin 4, type 1, R-cadherin (CDH4) NM_001794.2 Cadherin 5, type 2 (CDH5) NM_001795.3 Cadherin 6, type 2, K-cadherin (CDH6) NM_004932.2 Cadherin 7, type 2 (CDH7) NM_004361.2 NM_033646.1 canalicular multidrug resistance protein X96395.2 canalicular multispecific organic anion U63970.1 transporter (cMOAT) U49248.1 Ccanalicular multispecific organic anion AF083552.1 transporter 2 (CMOAT2) CD163 molecule-like 1 (CD163L1) NM_174941.4 CD4 molecule (CD4) NM_001195015.1 NM_001195017.1 NM_001195016.1 NM_001195014.1 NM_000616.4 CD47 molecule BC010016.2 BT006907.1 BC037306.1 BC012884.1 NM_198793.2 NM_001777.3 cellular proto-oncogene (c-mer) U08023.1 ceptor for advanced glycosylation end- AY755622.1 products intron 4 & 9 variant (AGER) Cholinergic receptor, nicotinic, alpha 1 NM_000079.3 (CHRNA1) NM_001039523.2 AK315312.1 Cholinergic receptor, nicotinic, alpha 10 NM_020402.2 (CHRNA10) Cholinergic receptor, nicotinic, alpha 2 BC153866.1 (CHRNA2) NM_000742.3 Cholinergic receptor, nicotinic, alpha 3 BC002996.1 (CHRNA3) BC098443.1 BC000513.2 BC001642.2 BC006114.1 NM_001166694.1 NM_000743.4 BT006897.1 BT006646.1 Cholinergic receptor, nicotinic, alpha 4 BC096293.3 GI:109731542 (CHRNA4) BC096290.1 BC096292.1 BC096291.1 NM_000744.5 AB209359.1 Cholinergic receptor, nicotinic, alpha 5 BC033639.1 (CHRNA5) NM_000745.3 Cholinergic receptor, nicotinic, alpha 6 BC014456.1 (CHRNA6) NM_001199279.1 NM_004198.3 AK313521.1 Cholinergic receptor, nicotinic, alpha 7 BC037571.1 (CHRNA7) NM_000746.4 NM_001190455.1 Cholinergic receptor, nicotinic, alpha 9 BC113549.1 (CHRNA9) BC113575.1 NM_017581.2 Cholinergic receptor, nicotinic, beta 1 BC023553.2 (CHRNB1) BC011371.1 NM_000747.2 Cholinergic receptor, nicotinic, beta 2 BC075041.2 (CHRNB2) BC075040.2 AK313470.1 NM_000748.2 Cholinergic receptor, nicotinic, beta 3 BC069788.1 (CHRNB3) BC069703.1 BC069681.1 NM_000749.3 Cholinergic receptor, nicotinic, beta 4 BC096080.1 BC096082.1 (CHRNB4) NM_000750.3 cholinergic receptor, nicotinic, delta BC093925.1 BC093923.1 (CHRND) NM_000751.1 Cholinergic receptor, nicotinic, epsilon NM_000080.3 (CHRNE) Cholinergic receptor, nicotinic, gamma BC111802.1 (CHRNG) NM_005199.4 CRB1 isoform II precursor AY043325.1 Cstic fibrosis transmembrane NM_000492.3 conductance regulator (ATP-binding cassette sub-family C, member 7) (CFTR) C-type lectin domain family 4, member NM_194450.2 A (CLEC4A) NM_194448.2 NM_194447.2 NM_016184.3 enaptin AF535142.1 Eph-related receptor transmembrane U62775.1 ligand Elk-L3 precursor (Elk-L3) Fc receptor related gene DQ021957.1 Fibroblast growth factor receptor 3 NM_022965.3 (FGFR3) Fibroblast growth factor receptor 4 NM_022963.2 (FGFR4) Fms-related tyrosine kinase 3 (FLT3) NM_004119.2 Follicle stimulating hormone receptor AY429104.1 (FSHR) S59900.1 M95489.1 M65085.1 BC118548.1 BC096831.1 BC125270.1 NM_181446.2 NM_000145.3 X68044.1 G protein-coupled receptor 155 BC035037.1 (GPR155) BC028730.1 NM_001033045.2 NM_152529.5 GABA-A receptor delta subunit AF016917.1 (GABRD) GABA-A receptor epsilon subunit U66661.1 GABAA receptor gamma 3 subunit S82769.1 GABA-A receptor pi subunit U95367.1 GABAA receptor subunit alpha4 U30461.1 GABA-A receptor theta subunit AF189259.1 (THETA) AF144648.1 GABA-A receptor, beta 1 subunit X14767.1 GABA-A receptor, gamma 2 subunit X15376.1 GABA-benzodiazepine receptor alpha- L08485.1 5-subunit (GABRA5) Gamma-aminobutyric acid (GABA) A BC030696.1 receptor, alpha 1 (GABRA1) NM_001127648.1 NM_001127647.1 NM_001127646.1 NM_001127645.1 NM_001127644.1 NM_001127643.1 NM_000806.5 Gamma-aminobutyric acid (GABA) A BC022488.1 receptor, alpha 2 (GABRA2) NM_001114175.1 NM_000807.2 Gamma-aminobutyric acid (GABA) A BC028629.1 receptor, alpha 3 (GABRA3) NM__000808.3 Gamma-aminobutyric acid (GABA) A BC035055.1 receptor, alpha 4 (GABRA4) NM_001204267.1 NM_001204266.1 NM_000809.3 Gamma-aminobutyric acid (GABA) A BC113422.1 receptor, alpha 5 (GABRA5) BC111979.1 BT009830.1 NM_001165037.1 NM_000810.3 Gamma-aminobutyric acid (GABA) A BC099641.3 receptor, alpha 6 (GABRA6) BC096241.3 BC099640.3 BC096242.3 NM_000811.2 Gamma-aminobutyric acid (GABA) A BC022449.1 receptor, beta 1 (GABRB1) NM_000812.3 Gamma-aminobutyric acid (GABA) A BC105639.1 receptor, beta 2 (GABRB2) BC099719.1 BC099705.1 NM_021911.2 NM_000813.2 gamma-aminobutyric acid (GABA) A BC010641.1 receptor, beta 3 (GABRB3) NM_001191320.1 NM_021912.4 NM_001191321.1 NM_000814.5 Gamma-aminobutyric acid (GABA) A BC033801.1 receptor, delta (GABRD) NM_000815.4 Gamma-aminobutyric acid (GABA) A BC059376.1 receptor, epsilon (GABRE) BC047108.1 BC026337.1 NM_004961.3 Gamma-aminobutyric acid (GABA) A BC031087.1 receptor, gamma 1 (GABRG1) NM_173536.3 Gamma-aminobutyric acid (GABA) A BC074795.2 GI:50959646 receptor, gamma 2 (GABRG2) BC059389.1 NM_198903.2 NM_000816.3 NM_198904.2 Gamma-aminobutyric acid (GABA) A NM_033223.4 receptor, gamma 3 (GABRG3) Gamma-aminobutyric acid (GABA) A BC074810.2 receptor, pi (GABRP) BC069348.1 BC074865.2 BC109105.1 BC109106.1 NM_014211.2 Gamma-aminobutyric acid (GABA) NM_002042.3 receptor, rho 1 (GABRR1) Gamma-aminobutyric acid (GABA) BC130352.1 receptor, rho 2 (GABRR2) BC130354.1 NM_002043.2 gamma-aminobutyric acid (GABA) NM_001105580.1 receptor, rho 3 (GABRR3) gamma-aminobutyric acid (GABA) BC109210.1 receptor, theta (GABRQ) BC109211.1 NM_018558.2 gamma-aminobutyric acid A receptor GU086164.1 beta 2 isoform 3 (GABRB2) GU086163.1 gamma-aminobutyric acid A receptor S67368.1 beta 2 subunit (GABR2) gamma-aminobutyric acid A receptor, AB209295.1 alpha 2 precursor gamma-aminobutyric acid receptor type M62400.1 A rho-1 subunit (GABA-A rho-1) gamma-aminobutyric acid type A S81944.1 receptor alpha 6 subunit gamma-aminobutyric acidA receptor S62907.1 alpha 2 subunit gamma-aminobutyric acidA receptor S62908.1 alpha 3 subunit gamma-aminobutyric-acid receptor X13584.1 alpha-subunit glycine receptor alpha 3 subunit U93917.1 glycine receptor alpha2 subunit B AY437084.1 AY437083.1 (GLRA2) glycine receptor beta subunit precursor AF094755.1 AF094754.1 (GLRB) Glycine receptor, alpha 1 (GLRA1) BC114967.1 BC114947.1 BC074980.2 NM_001146040.1 NM_000171.3 Glycine receptor, alpha 2 (GLRA2) BC032864.2 NM_001171942.1 NM_001118886.1 NM_001118885.1 NM_002063.3 Glycine receptor, alpha 3 (GLRA3) BC036086.1 NM_006529.2 NM_001042543.1 Glycine receptor, alpha 4 (GLRA4) NM_001172285.1 NM_001024452.2 glycine receptor, beta (GLRB) BC032635.1 NM_001166061.1 NM_000824.4 NM_001166060.1 GP2 D38225.1 gpVI mRNA for platelet glycoprotein VI AB035073.1 H1 histamine receptor Z34897.1 HEK2 protein tyrosine kinase receptor X75208.1 high affinity IgE receptor alpha-subunit X06948.1 (FcERI) HLA D32131.1 D32129.1 HLA class I locus C heavy chain X58536.1 HLA class II DR-beta (HLA-DR B) X12544.1 HLA classII histocompatibility antigen X00452.1 alpha-chain HLA-A26 (HLA class-I heavy chain) D32130.1 HLA-DR antigens associated invariant X00497.1 chain (p33) holinergic receptor, nicotinic, delta AK315297.1 polypeptide(CHRND) HPTP (protein tyrosine phosphatase X54133.1 delta) HPTP (protein tyrosine phosphatase X54134.1 epsilon) HPTP (protein tyrosine phosphatase X54135.1 zeta) HPTP alpha mRNA for protein tyrosine X54130.1 phosphatase alpha HPTP beta (protein tyrosine phosphatase X54131.1 beta) -hydroxytryptamine (serotonin) receptor NM_001163646.1 3 family member D (HTR3D) ICAM-3 X69819.1 IL-12 receptor component U03187.1 IL-4-R X52425.1 immunoglobulin receptor precursor AY046418.1 insulin-like growth factor I receptor X04434.1 integrin associated protein Z25521.1 Killer cell lectin-like receptor subfamily NM_001114396.1 D, member 1 (KLRD1) KIR (cl-11) NK receptor precursor U30274.1 protein U30273.1 U30272.1 large conductance calcium- and voltage- U11058.2 dependent potassium channel alpha subunit (MaxiK) large-conductance calcium-activated AF160967.1 potassium channel beta subunit (KCNMB4) leucine-rich glioma-inactivated protein AF055636.1 precursor (LGI1) Leukocyte immunoglobulin-like NM_001130917.1 receptor, subfamily A (with TM NM_006866.2 domain), member 2 (LILRA2) Leukocyte immunoglobulin-like NM_006865.3 receptor, subfamily A (without TM NM_001172654.1 domain), member 3 (LILRA3) lycine receptor beta subunit (GLRB) U33267.1 lymphocte activation marker Blast-1 X06341.1 M-ABC2 protein (M-ABC2), nuclear AF216833.1 gene for mitochondrial product Major histocompatibility complex, class NM_002116.6 I, A (HLA-A) Major histocompatibility complex, class NM_005514.6 I, B (HLA-B) Major histocompatibility complex, class NM_002117.4 I, C (HLA-C) Major histocompatibility complex, class NM_005516.5 I, E (HLA-E) Major histocompatibility complex, class NM_002127.5 I, G (HLA-G), MAT8 protein X93036.1 MCTP1L mRNA AY656715.1 MCTP1S AY656716.1 MCTP2 AY656717.1 membrane glycoprotein P (mdr3) M23234.1 Mint1 AF029106.1 mono ATP-binding cassette protein AB013380.1 GI:12248754 MRP5 AB019002.1 MRP6 (MRP6) AF076622.1 MT-ABC transporter (MTABC) AF076775.1 multidrug resistance protein 1 EU854148.1 EU852583.1 AB208970.1 multidrug resistance protein 3 (ABCC3) Y17151.2 multidrug resistance protein 5 (MRP5) U83661.2 multidrug resistance-associated protein AY081219.1 (ABCC4) multidrug resistance-associated protein L05628.1 (MRP) multidrug resistance-associated protein 3 AF085690.1 (MRP3) AF085691.1 Multidrug resistance-associated protein AB209454.1 5 variant protein multidrug resistance-associated protein 7 AY032599.1 (SIMRP7) multidrug resistance-associated protein AF009670.1 homolog MRP3 (MRP3) multidrug resistance-associated AB010887.1 protein(MRP)-like protein-2 (MLP-2) multiple C2 domains, transmembrane 1 BC030005.2 (MCTP1) NM_001002796.2 NM_024717.4 multiple C2 domains, transmembrane 2 BC111024.1 (MCTP2) BC041387.1 BC025708.1 BC131527.1 NM_001159644.1 NM_018349.3 NM_001159643.1 myeloid cell leukemia ES variant FJ917536.1 (MCL1) neuregulin 4 (NRG4) AM392365.1 AM392366.1 neuronal nAChR beta-3 subunit X67513.1 neuronal nicotinic acetylcholine alpha10 AJ278118.1 subunit (NACHRA10 gene) AJ295237.1 neuronal nicotinic acetylcholine receptor X53559.1 alpha-3 subunit nicotinic acetylcholine alpha-7 subunit X70297.1 AJ586911.1 (CHRNA7 gene) neuronal nicotinic acetylcholine receptor X53179.1 beta-2 subunit nicotinic acetylcholine receptor alpha 3 M86383.1 subunit precursor nicotinic acetylcholine receptor alpha 4 L35901.1 subunit (nAChR) nicotinic acetylcholine receptor alpha 9 AJ243342.1 subunit (NACHRA9 gene) nicotinic acetylcholine receptor alpha2 U62431.1 subunit precursor Y16281.1 nicotinic acetylcholine receptor alpha3 U62432.1 subunit precursor Y08418.1 nicotinic acetylcholine receptor alpha4 U62433.1 subunit precursor Y08421.1 X87629.1 nicotinic acetylcholine receptor alpha5 U62434.1 subunit precursor Y08419.1 nicotinic acetylcholine receptor alpha6 U62435.1 subunit precursor Y16282.1 nicotinic acetylcholine receptor alpha7 U62436.1 subunit precursor nicotinic acetylcholine receptor alpha7 Y08420.1 subunit precursor nicotinic acetylcholine receptor beta2 U62437.1 subunit precursor nicotinic acetylcholine receptor beta2 Y08415.1 subunit precursor nicotinic acetylcholine receptor beta3 U62438.1 subunit precursor nicotinic acetylcholine receptor beta3 Y08417.1 subunit precursor nicotinic acetylcholine receptor beta4 U62439.1 subunit precursor nicotinic acetylcholine receptor beta4 Y08416.1 subunit precursor nicotinic acetylcholine receptor subunit AF199235.2 alpha 10 nicotinic cholinergic receptor alpha 7 AF385585.1 (CHRNA7) nicotinic receptor alpha 5 subunit M83712.1 nicotinic receptor beta 4 subunit X68275.1 on-erythroid band 3-like protein (HKB3) X03918.1 p58 natural killer cell receptor precursor U24079.1 U24078.1 U24077.1 U24076.1 U24075.1 U24074.1 peptide transporter (TAP1) L21207.1 L21206.1 L21205.1 L21204.1 peroxisomal 70 kD membrane protein M81182.1 peroxisomal membrane protein 69 AF009746.1 (PMP69) P-glycoprotein AY090613.1 P-glycoprotein (ABCB1) AF399931.1 AF319622.1 P-glycoprotein (mdr1) AF016535.1 P-glycoprotein (PGY1) M14758.1 P-glycoprotein ABCB5 AY234788.1 Phospholipase A2 receptor 1, 180 kDa NM_001007267.2 (PLA2R1) PMP70 X58528.1 Potassium voltage-gated channel, NM_002234.2 shaker-related subfamily, member 5 (KCNA5) potassium voltage-gated channel, NM_031886.2 shaker-related subfamily, member 7 (KCNA7) precursor of epidermal growth factor X00588.1 receptor pre-T cell receptor alpha-type chain U36759.1 precursor protein tyrosine phosphatase hPTP-J U73727.1 precursor Protein tyrosine phosphatase, receptor NM_006504.4 type, F (PTPRF) 2 NM_130435.3 NM_002840.3 NM_130440.2 Protein tyrosine phosphatase, receptor NM_002841.3 type, G (PTPRG) Protein tyrosine phosphatase, receptor NM_001161440.1 type, H (PTPRH) NM_002842.3 Protein tyrosine phosphatase, receptor NM_002843.3 type, J (PTPRJ) NM_001098503.1 Protein tyrosine phosphatase, receptor NM_001135648.1 type, K (PTPRK) NM_002844.3 Protein tyrosine phosphatase, receptor NM_001105244.1 type, M (PTPRM) NM_002845.3 Protein tyrosine phosphatase, receptor NM_001199764.1 type, N polypeptide 2 (PTPRN2) NM_002846.3 NM_001199763.1 NM_130843.2 NM_002847.3 NM_130842.2 Protein tyrosine phosphatase, receptor NM_130846.1 type, R (PTPRR) NM_002849.2 Protein tyrosine phosphatase, receptor NM_007050.5 type, T (PTPRT) NM_133170.3 Protein tyrosine phosphatase, receptor NM_001195001.1 type, U (PTPRU) NM_133178.3 protein tyrosine phosphatase, receptor NM_005704.4 type, U (PTPRU) NM_133177.3 protocadherin 1 (PCDH1) NM_002587.3 NM_032420.2 Protocadherin 8 (PCDH8), transcript NM_032949.2 variant 2 NM_002590.3 Protocadherin 9 (PCDH9) NM_203487.2 protocadherin alpha 1 (PCDHA1) NM_020403.4 NM_031411.1 Protocadherin alpha 10 (PCDHA10) NM_031860.1 protocadherin alpha 6 (PCDHA6) NM_031849.1 protocadherin gamma subfamily A, 1 NM_018912.2 (PCDHGA1) NM_031993.1 protocadherin gamma subfamily A, 10 NM_018913.2 (PCDHGA10) NM_032090.1 Protocadherin gamma subfamily A, 11 NM_032092.1 (PCDHGA11) NM_032091.1 NM_018914.2 Protocadherin gamma subfamily A, 12 NM_032094.1 (PCDHGA12) NM_003735.2 Protocadherin gamma subfamily A, 2 NM_032009.1 (PCDHGA2) NM_018915.2 protocadherin gamma subfamily A, 3 NM_018916.3 (PCDHGA3) protocadherin gamma subfamily A, 3 NM_032011.1 (PCDHGA3) protocadherin gamma subfamily A, 4 NM_032053.1 (PCDHGA4) NM_018917.2 protocadherin gamma subfamily A, 5 NM_032054.1 (PCDHGA5) NM_018918.2 protocadherin gamma subfamily A, 6 NM_032086.1 (PCDHGA6), transcript variant 2 NM_018919.2 protocadherin gamma subfamily A, 7 NM_018920.2 (PCDHGA7) NM_032087.1 Protocadherin gamma subfamily A, 8 NM_032088.1 (PCDHGA8) NM_014004.2 protocadherin gamma subfamily A, 9 NM_018921.2 (PCDHGA9) NM_032089.1 protocadherin gamma subfamily B, 1 NM_018922.2 (PCDHGB1) NM_032095.1 protocadherin gamma subfamily B, 2 NM_018923.2 (PCDHGB2) NM_032096.1 protocadherin gamma subfamily B, 3 NM_018924.2 (PCDHGB3) NM_032097.1 Protocadherin gamma subfamily B, 4 NM_032098.1 (PCDHGB4) NM_003736.2 protocadherin gamma subfamily B, 5 NM_032099.1 (PCDHGB5) NM_018925.2 protocadherin gamma subfamily B, 6 NM_032100.1 (PCDHGB6) NM_018926.2 Protocadherin gamma subfamily B, 7 NM_032101.1 (PCDHGB7) NM_018927.2 Protocadherin gamma subfamily C, 3 NM_032403.1 (PCDHGC3) NM_032402.1 NM_002588.2 protocadherin gamma subfamily C, 4 NM_018928.2 (PCDHGC4) NM032406.1 protocadherin gamma subfamily C, 5 NM_032407.1 (PCDHGC5) NM_018929.2 PSF-2 M74447.1 transmembrane receptor IL-1Rrp U43672.1 RING4 X57522.1 Sarcoglycan, zeta (SGCZ) NM_139167.2 SB classII histocompatibility antigen X00457.1 alpha-chain SH2 domain-containing phosphatase AF319440.1 anchor protein 1c (SPAP1) SMRP AB005659.1 Solute carrier family 4, sodium NM_001134742.1 bicarbonate cotransporter, member 4 NM_003759.3 (SLC4A4) NM_001098484.2 Solute carrier family 6 (neurotransmitter NM_001172504.1 transporter, noradrenalin), member 2 NM_001172502.1 (SLC6A2) NM_001172501.1 NM_001043.3 sulfonylurea receptor (SUR1) U63421.1 AB209084.1 AF087138.1 sushi-repeat-containing protein U78093.1 precursor (SRPX) Synaptotagmin XIII (5YT13) NM_020826.2 Synaptotagmin XV (5YT15) NM_031912.4 NM_181519.2 T200 leukocyte common antigen Y00062.1 (CD45, LC-A) TAP2B Z22935.1 TAP2E Z22936.1 TAPL (TAP-Like), AB112583.1 AB112582.1 AB045381.2 thyroperoxidase Y00406.1 tissue-type tonsil IFGP6 AY212514.1 trans-golgi network glycoprotein 48 AF027515.1 (TGN) trans-golgi network glycoprotein 51 AF027516.1 (TGN) Transporter 1, ATP-binding cassette, BC014081.2 sub-family B (MDR/TAP) (TAP1) NM_000593.5 AY523971.2 AY523970.1 Transporter 2, ATP-binding cassette, AF078671.1 AF105151.1 sub-family B (MDR/TAP) (TAP2), NM_018833.2 NM_000544.3 AK223300.1 AK222823.1 AB073779.1 AB208953.1 ATP-binding cassette transporter sub- AY344117.1 family C member 13 (ABCC13) tyrosine kinase (FER) J03358.1 Ubiquinol-cytochrome c reductase, NM_006003.2 Rieske iron-sulfur polypeptide 1 (UQCRFS1) BC067832.1 BC010035.2 BC000649.1 ulfonylurea receptor (SUR1) L78207.1 Cell-Type Specific Polypeptides

As used herein, the term “cell-type specific polypeptide” refers to a polypeptide that is expressed in a cell having a particular phenotype (e.g., a muscle cell) but is not generally expressed in other cell types with different phenotypes. For example, MyoD is expressed specifically in muscle cells but not in non-muscle cells, thus MyoD is a cell-type specific polypeptide. As another example, albumin is expressed in hepatocytes and is thus an hepatocyte-specific polypeptide.

Such cell-specific polypeptides are well known in the art or can be found using a gene array analysis and comparison of at least two different cell types. Methods for gene expressional array analysis is well known in the art.

Differentiation factors, reprogramming factors and transdifferentiation factors are further discussed herein in their appropriate sub-sections.

Death Receptors and Death Receptor Ligands

By “death receptor” is meant a receptor that induces cellular apoptosis once bound by a ligand. Death receptors include, for example, tumor necrosis factor (TNF) receptor superfamily members having death domains (e.g., TNFRI, Fas, DR3, 4, 5, 6) and TNF receptor superfamily members without death domains LTbetaR, CD40, CD27, HVEM. Death receptors and death receptor ligands are well known in the art or are discussed herein.

The synthetic, modified RNAs described herein can encode for death receptors to be expressed on the surface of a cell to enhance the vulnerability of a cell to apoptosis. The death ligand can also be encoded or can be provided e.g., at a tumor site. This is particularly useful in the treatment of cancer, where cells evade apoptosis and continue to divide. Alternatively, the synthetic, modified RNAs or compositions thereof can encode for a death receptor ligand, which will induce apoptosis in cells that express a cell surface death receptor and can increase the efficiency of programmed cell death in targeted cells of a subject.

Some non-limiting examples of death receptors include FAS (CD95, Apo1), TNFR1 (p55, CD120a), DR3 (Apo3, WSL-1, TRAMP, LARD), DR4, DR5 (Apo2, TRAIL-R2, TRICK2, KILLER), CAR1, and the adaptor molecules FADD, TRADD, and DAXX. Some non-limiting examples of death receptor ligands include FASL (CD95L), TNF, lymphotoxin alpha, Apo3L (TWEAK), and TRAIL (Apo2L).

Mitogen Receptors

The synthetic, modified RNAs described herein can be used to express a mitogen receptor on a cell surface. Activation of a mitogen receptor with the mitogen induces cell growth and/or differentiation of the cell.

Mitogen receptors include those that bind ligands including, but not limited to: insulin, insulin-like growth factor (e.g., IGF1, IGF2), platelet derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), fibroblast growth factor (FGF), bone morphogenic proteins (BMPs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha and -beta, among others.

In addition, cytokines that promote cell growth can also be encoded by synthetic, modified RNAs herein. For example, cytokines such as erythropoietin, thrombopoietin and other cytokines from the IL-2 sub-family tend to induce cell proliferation and growth.

Protein Therapeutics

Synthetic, modified RNAs as described herein can also be used to express protein therapeutically in cells by either administration of a synthetic, modified RNA composition to an individual or by administering a synthetic, modified RNA to cells that are then introduced to an individual. In one aspect, cells can be transfected with a modified RNA to express a therapeutic protein using an ex vivo approach in which cells are removed from a patient, transfected by e.g., electroporation or lipofection, and re-introduced to the patient. Continuous or prolonged administration in this manner can be achieved by electroporation of blood cells that are re-infused to the patient.

Some exemplary protein therapeutics include, but are not limited to: insulin, growth hormone, erythropoietin, granulocyte colony-stimulating factor (G-CSF), thrombopoietin, clotting factor VII, Factor IX, interferon, glucocerebrosidase, anti-HER2 monoclonal antibody, and Etanercept, among others.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. In addition, the term ‘cell’ can be construed as a cell population, which can be either heterogeneous or homogeneous in nature, and can also refer to an aggregate of cells.

It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

All references cited herein in the specification are incorporated by reference in their entirety.

EXAMPLES

Currently, clinical applications using induced pluripotent stem (iPS) cells are impeded by low efficiency of iPS derivation, and the use of protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe, reliable, and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Described herein are novel, non-mutagenic strategies for altering cellular phenotypes, such as reprogramming cell fate, based on the administration of synthetic, modified mRNAs that are modified to overcome innate cellular anti-viral responses. The compositions and approaches described herein can be used to reprogram multiple human cell types to pluripotency with surprising and unexpected efficiencies that greatly surpass established protocols. Also described herein are novel compositions and methods for directing the fate of cells towards clinically useful cell types, and a non-limiting example that demonstrates that this technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Thus, the compositions and methods described herein represent safe, highly efficient strategies for altering cellular developmental potentials, such as somatic cell reprogramming and directing differentiated cell fates, that have broad applicability for basic research, disease modeling and regenerative and personalized medicine.

Experimental Procedures

Construction of IVT Templates

The pipeline for production of IVT template constructs and subsequent RNA synthesis is schematized in FIG. 1. The oligonucleotide sequences used in the construction of IVT templates are shown in Table 4. All oligos were synthesized by Integrated DNA Technologies (Coralville, Iowa). ORF PCRs were templated from plasmids bearing human KLF4, c-MYC, OCT4, SOX2, human ES cDNA (LIN28), Clontech pIRES-eGFP (eGFP), pRVGP (d2eGFP) and CMV-MyoD from Addgene. The ORF of the low-stability nuclear GFP was constructed by combining the d2eGFP ORF with a 3′ nuclear localization sequence. PCR reactions were performed using HiFi Hotstart (KAPA Biosystems, Woburn, Mass.) per the manufacturer's instructions. Splint-mediated ligations were carried out using Ampligase Thermostable DNA Ligase (Epicenter Biotechnologies, Madison, Wis.). UTR ligations were conducted in the presence of 200 nM UTR oligos and 100 nM splint oligos, using 5 cycles of the following annealing profile: 95° C. for 10 seconds; 45° C. for 1 minute; 50° C. for 1 minute; 55° C. for 1 minute; 60° C. for 1 minute. A phosphorylated forward primer was employed in the ORF PCRs to facilitate ligation of the top strand to the 5′ UTR fragment. The 3′ UTR fragment was also 5′-phosphorylated using polynucleotide kinase (New England Biolabs, Ipswich, Mass.). All intermediate PCR and ligation products were purified using QIAquick spin columns (Qiagen, Valencia, Calif.) before further processing. Template PCR amplicons were sub-cloned using the pcDNA 3.3-TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.). Plasmid inserts were excised by restriction digest and recovered with SizeSelect gels (Invitrogen) before being used to template tail PCRs.

5′ and 3′ UTR oligos are ligated to the top strand of gene-specific ORF amplicons to produce a basic template construct for cloning. Underlined bases in the 5′ UTR oligo sequence indicate the upstream T7 promoter, and in the 3′ UTR oligo sequence show downstream restriction sites, introduced to facilitate linearization of template plasmids. Template PCR primers are used to amplify ligation products for sub-cloning. Tail PCR primers are used to append an oligo(dT) sequence immediately after the 3′ UTR to drive templated addition of a poly(A) tail during IVT reactions. Gene-specific ORF primers are used to capture the coding region (minus the start codon) from cDNA templates. Splint oligos mediate ligation of UTR oligos to the top strand of ORF amplicons.

TABLE 4 Oligonucleotides for IVT template construction (SEQ ID NOs: 1429-1466, respectively, in order of appearance) ORF Forward Primer ORF Reverse Primer eGFP GTGAGCAAGGGCGAGGAGCTGTT TTACTTGTACAGCTCGTCCATGCCGAGA D2eGFP GTGAGCAAGGGCGAGGAGCTGTT CTACACATTGATCCTAGCAGAAGCACAGGCT K1F4 GCTGTCAGCGACGCGCTGCTC TTAAAAATGCCTCTTCATGTGTAAGGCGAGGT c-MYC CCCCTCAACGTTAGCTTCACCAATTTC TTACGCACAAGAGTTCCGTAGCTGTTCA OCT4 GCGGGACACCTGGCTTCGGATTC TCAGTTTGAATGCATGGGAGAGCCCAGA SOX2 TACAACATGATGGAGACGGAGCTGAAGC TCACATGTGTGAGAGGGGCAGTGTG LIN28 GGCTCCGTGTCCAACCAG TCAATTCTGTGCCTCCGG MYOD GAGCTTCTATCGCCGCCACTCC TCAAAGCACCTGATAAATCGATTGG 5′ Splint Oligo 3′ Splint Oligo eGFP TCCTCGCCCTTGCTCACCATGGGGTTTATATTTCTTCTT CCCGCAGAAGGCAGCTTACTTGTACAGCTCGTCCATGC D2eGFP TCCTCGCCCTTGCTCACCATGGGGTTTATATTTCTTCTT CCCGCAGAAGGCAGCCTACACATTGATCCTAGCAGA K1F4 GCGCGTCGCTGACAGCCATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTTAAAAATGCCTCTTCATGTGTAA c-MYC GTGAAGCTAACGTTGAGGGGCATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTTACGCACAAGAGTTCCGTAG OCT4 AAGCCAGGTGTCCCGCCATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTCAGTTTGAATGCATGGGAG SOX2 CTCCGTCTCCATCATGTTGTACATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTCACATGTGTGAGAGGGGC LIN28 CTGGTTGGACACGGAGCCCATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTCAATTCTGTGCCTCCGG MYOD TGGCGGCGATAGAAGCTCCATGGTGGCTCTTATATTTCTTCTT CCCGCAGAAGGCAGCTCAAGCACCTGATAAATCGCATTGG UTR Oligos 5' UTR TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATG 3' UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAGTGA Forward Primer Reverse Primer Template TTGGACCCTCGTACAGAAGCTAATACG GCGTCGACACTAGTTCTAGACCCTCA PCR Tail PCR TTGGACCCTCGTACAGAAGCTAATACG T₁₂₀CTTCCTACTCAGGCTTTATTCAAAGACCA Synthesis of Synthetic, Modified RNA

RNA was synthesized with the MEGAscript T7 kit (Ambion, Austin, Tex.), using 1.6 ug of purified tail PCR product to template each 40 uL reaction. A custom ribonucleoside blend was used comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs), adenosine triphosphate and guanosine triphosphate (USB, Cleveland, Ohio), 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, Calif.). Final nucleotide reaction concentrations were 33.3 mM for the cap analog, 3.8 mM for guanosine triphosphate, and 18.8 mM for the other nucleotides. Reactions were incubated 3-6 hours at 37° C. and DNAse-treated as directed by the manufacturer. RNA was purified using Ambion MEGAclear spin columns, then treated with Antarctic Phosphatase (New England Biolabs) for 30 minutes at 37° C. to remove residual 5′-triphosphates. Treated RNA was re-purified, quantitated by Nanodrop (Thermo Scientific, Waltham, Mass.), and adjusted to 100 ng/uL working concentration by addition of Tris-EDTA (pH 7.0). RNA reprogramming cocktails were prepared by pooling individual 100 ng/uL RNA stocks to produce a 100 ng/uL (total) blend. The KMOS[L]+GFP cocktails were formulated to give equal molarity for each component except for OCT4, which was included at 3× molar concentration. Volumetric ratios used for pooling were as follows: 170:160:420:130:120[:90] (KLF4:c-MYC:OCT4: SOX2:GFP[:LIN28]).

Cells

The following primary cells were obtained from ATCC (Manassas, Va.): human neonatal epidermal keratinocytes, BJ human neonatal foreskin fibroblasts, MRC-5 human fetal lung fibroblasts, and Detroit 551 human fetal skin fibroblasts. CF cells were obtained with informed consent from a skin biopsy taken from an adult cystic fibrosis patient. The Daley Lab provided dH1f fibroblasts, which were sub-cloned from fibroblasts produced by directed differentiation of the H1-OGN human ES cell line as previously described (Park et al., 2008). BGO1 hES cells were obtained from BresaGen (Athens, Ga.). H1 and H9 hES cells were obtained from WiCell (Madison, Wi).

RNA Transfection

RNA transfections were carried out using RNAiMAX (Invitrogen) or TransIT-mRNA (Mirus Bio, Madison, Wis.) cationic lipid delivery vehicles. RNAiMAX was used for RiPS derivations, the RiPS-to-myogenic conversion, and for the multiple cell-type transfection experiment documented in FIGS. 3A-3F. All other transfections were performed with TransIT-mRNA. For RNAiMAX transfections, RNA and reagent were first diluted in Opti-MEM basal media (Invitrogen). 100 ng/uL RNA was diluted 5× and 5 uL of RNAiMAX per microgram of RNA was diluted 10×, then these components were pooled and incubated 15 minutes at room temperature before being dispensed to culture media. For TransIT-mRNA transfections, 100 ng/uL RNA was diluted 10× in Opti-MEM and BOOST reagent was added (2 uL per microgram of RNA), then TransIT-mRNA was added (2 uL per microgram of RNA), and the RNA-lipid complexes were delivered to culture media after a 2-minute incubation at room temperature. RNA transfections were performed in Nutristem xeno-free hES media (Stemgent, Cambridge, Mass.) for RiPS derivations, Dermal Cell Basal Medium plus Keratinocyte Growth Kit (ATCC) for keratinocyte experiments, and Opti-MEM plus 2% FBS for all other experiments described. The B18R interferon inhibitor (eBioscience, San Diego, Calif.) was used as a media supplement at 200 ng/mL.

qRT-PCR of Interferon-Regulated Genes

Transfected and control 6-well cultures were washed with PBS and lysed in situ using 400 uL CellsDirect resuspension buffer/lysis enhancer (Invitrogen) per well, and 20 uL of each lysate was taken forward to a 50 uL reverse transcription reaction using the VILO cDNA synthesis kit (Invitrogen). Completed reactions were purified on QIAquick columns (Qiagen), and analyzed in 20 uL qPCRs, each templated with ˜10% of the total cDNA prep. The reactions were performed using SYBR FAST qPCR supermix (KAPA Biosystems) with 250 nM primers and a thermal profile including 35 cycles of (95° C. 3 s; 60° C. 20 s). The qPCR primer sequences used are given Table 5.

TABLE 5 Primers for qRT-PCR analysis of interferon-regulated genes (SEQ ID NOs: 1467-1480, respectively, in order of appearance). Transcript Forward Primer Reverse Primer GAPDH GAAGGCTGGGGCTCATTT CAGGAGGCATTGCTGATGAT IFNA ACCCACAGCCTGGATAACAG ACTGGTTGCCATCAAACTCC IFNB CATTACCTGAAGGCCAAGGA CAGCATCTGCTGGTTGAAGA IFIT1 AAAAGCCCACATTTGAGGTG GAAATTCCTGAAACCGACCA OAS1 CGATCCCAGGAGGTATCAGA TCCAGTCCTCTTCTGCCTGT PKR TCGCTGGTATCACTCGTCTG GATTCTGAAGACCGCCAGAG RIG-I GTTGTCCCCATGCTGTTCTT GCAAGTCTTACATGGCAGCA Reprogramming to Pluripotency

Gamma-irradiated human neonatal fibroblast feeders (GlobalStem, Rockville, Md.) were seeded at 33,000 cells/cm2. Nutristem media was used during the reprogramming phase of these experiments. Media was replaced daily, four hours after transfection, and supplemented with 100 ng/mL bFGF (Stemgent) and 200 ng/mL B18R before use. Where applied, VPA was added to media at 1 mM final concentration on days 8-15 of reprogramming. Low-oxygen culture experiments were carried out in a NAPCO 8000 WJ incubator (Thermo Scientific) supplied by NF300 compressed nitrogen cylinders (Airgas, Radnor, Pa.). Media were equilibrated at 5% O₂ for approximately 4 hours before use. Cultures were passaged using TrypLE Select recombinant protease (Invitrogen). Y27632 ROCK inhibitor (Watanabe et al., 2007) was purchased from Stemgent and included at 10 uM in recipient plates until the next media change, except where otherwise indicated. The daily RNA dose applied in the RiPS derivations was 1200 ng per well (6-well plate format) or 8 ug to a 10-cm dish.

For the RNA vs. retrovirus trial, both arms of the experiment were started with the same number of dH1f cells, and the passaging of the cultures was synchronized. Starting cultures were seeded with 100,000 cells in individual wells of a 6-well plate using fibroblast media (DMEM+10% FBS). The following day (day 1) KMOS RNA transfections were initiated in the RNA plate, and the viral plate was transduced with a KMOS retroviral cocktail (MOI=5 for each virus). All wells were passaged on day 6, using split ratios of 1:6 for the RNA wells and 1:3 for the virus wells. The conditions applied in the RNA arm of the trial were as in the initial RiPS derivation, including the use of Nutristem supplemented with 100 ng/mL bFGF, 5% O2 culture, and human fibroblast feeders. Ambient oxygen tension and other conventional iPS derivation conditions were used in the viral arm, the cells being grown in fibroblast media without feeders until the day 6 split, then being replated onto CF1 MEF feeders (GlobalStem) with a switch to hES media based on Knockout Serum Replacement (Invitrogen) supplemented with 10 ng/mL bFGF.

Culture of RIPS Cell Colonies

Emerging RiPS cell colonies were picked and clonally transferred to MEF-coated 24-well plates (Nunc, Rochester, N.Y.) with standard hES medium containing 5 uM Y27632 (BioMol, Plymouth Meeting, Pa.). The hES media comprised DMEM/F12 supplemented with 20% Knockout Serum Replacement (Invitrogen), 10 ng/mL of bFGF (Gembio, West Sacramento, Calif.), lx non-essential amino acids (Invitrogen), 0.1 mM β-ME (Sigma), 1 mM L-glutamine (Invitrogen), plus antibiotics. Clones were mechanically passaged once more to MEF-coated 6-well plates (Nunc), and then expanded using enzymatic passaging with collagenase IV (Invitrogen). For RNA and DNA preparation, cells were plated onto hES-qualified Matrigel (BD Biosciences) in mTeSR (Stem Cell Technologies, Vancouver, BC), and further expanded by enzymatic passaging using dispase (Stem Cell Technologies).

Immunostaining of Pluripotency Markers

For fixed-cell imaging, RiPS colonies were mechanically picked and plated onto MEF feeders in black 96-well plates (Matrix Technologies, Maumee, Ohio). Two days post-plating, cells were washed with PBS and fixed in 4% paraformaldehyde for 20 minutes. After 3 PBS washes, cells were treated with 0.2% Triton X (Sigma) in PBS for 30 minutes to allow nuclear permeation. Cells were washed 3× in PBS and blocked in blocking buffer containing 3% BSA (Invitrogen) and 5% donkey serum (Sigma) for 2 hours at room temperature. After three PBS washes, cells were stained in blocking buffer with primary and conjugated antibodies at 4° C. overnight. After washing 3× with PBS, cells were stained with secondary antibodies and 1 ug/mL Hoechst 33342 (Invitrogen) in blocking buffer for 3 hours at 4° C. or for 1 hour at room temperature, protected from light. Cells were washed 3× with PBS before visualization. The following antibodies were used, at 1:100 dilution: TRA-1-60-Alexa Fluor 647, TRA-1-81-Alexa Fluor 488, SSEA-4-Alexa Fluor 647, and SSEA-3-Alexa 488 (BD Biosciences). Primary OCT4 and NANOG antibodies (Abcam, Cambridge, Mass.) were used at 0.5 ug/mL, and an anti-rabbit IgG Alexa Fluor 555 (Invitrogen) was used as the secondary. Images were acquired with a Pathway 435 bioimager (BD Biosciences) using a 10× objective. Live imaging was performed as described previously (Chan et al., 2009). Briefly, wells were stained by adding 1:100-diluted TRA-1-60-Alexa 647 and SSEA-4-Alexa 555 antibodies (BD Biosciences) to culture media. After 1.5 hours, Hoechst 33342 was added at a final concentration of 0.25 ug/mL, and wells were incubated for an additional 30 minutes. Wells were washed 3× with DMEM/F12 base media lacking phenol red, and imaged in hES media lacking phenol red. Images were acquired with a Pathway 435 bioimager using 4× and 10× objectives. Post-acquisition image processing and analysis was performed using Adobe Photoshop for pseudocoloring and ImageJ (http://rsbweb.nih.gov/ij) for flat-field correction, background subtraction, and colony quantitation.

For pluripotency factor time course experiments, transfected human epidermal keratinocytes were trypsinized, washed with PBS, and fixed in 4% paraformaldehyde for 10 minutes. Fixed cells were washed with 0.1M glycine, then blocked and permeabilized in PBS/0.5% saponin/1% goat serum (Rockland Immunochemicals, Gilbertsville, Pa.) for 20 minutes. Cells were incubated for 1 hour at room temperature with 1:100 diluted primary antibodies for KLF4, OCT4, SOX2 (Stemgent), washed, then for 45 minutes at room temperature with 1:200-diluted DyLight 488-labeled secondary antibodies (goat anti-mouse IgG+IgM and goat anti-rabbit IgG). Cells suspended in PBS were analyzed by flow cytometry.

Gene Expression Analysis

RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. First-strand cDNA was primed with oligo(dT) primers and qPCR was performed with primer sets as described previously (Park et al., 2008), using Brilliant SYBR Green master mix (Stratagene, La Jolla, Calif.). For the microarray analysis, RNA probes were prepared and hybridized to Human Genome U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, Calif.) per the manufacturer's instructions. Arrays were processed by the Coriell Institute Genotyping and Microarray Center (Camden, N.J.). Microarray data will be uploaded to the GEO database. Gene expression levels were normalized with the Robust Multichip Average (RMA) algorithm. Unsupervised hierarchical clustering was performed using the Euclidean distance with average linkage method. The similarity metric for comparison between different cell lines is indicated on the height of cluster dendrogram.

Bisulfite Sequencing

DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol. Bisulfite treatment of genomic DNA was carried out using EZ DNA Methylation™ Kit (Zymo Research, Orange, Calif.) according to the manufacturer's protocol. For pyrosequencing analysis, the bisulfate treated DNA was first amplified by HotStar Taq Polymerase (Qiagen) for 45 cycles of (95° C. 30 s; 53° C. 30 s; 72° C. 30 s). The analysis was performed by EpigenDx using the PSQ™96HS system according to standard procedures using primers that were developed by EpigenDx for the CpG sites at positions (−50) to (+96) from the start codon of the OCT4 gene.

Tri-Lineage Differentiation

Embryoid body (EB) hematopoietic differentiation was performed as previously described (Chadwick et al., 2003). Briefly, RiPS cells and hES cell controls were passaged with collagenase IV and transferred (3:1) in differentiation medium to 6-well low-attachment plates and placed on a shaker in a 37° C. incubator overnight. Starting the next day, media was supplemented with the following hematopoietic cytokines: 10 ng/mL of interleukin-3 (R&D Systems, Minneapolis, Minn.) and interleukin-6 (R&D), 50 ng/mL of G-CSF (Amgen, Thousand Oaks, Calif.) and BMP-4 (R&D), and 300 ng/mL of SCF (Amgen) and Flt-3 (R&D). Media was changed every 3 days. On day 14 of differentiation, EBs were dissociated with collagenase B (Roche, Indianapolis, Ind.). 2×104 differentiated cells were plated into methylcellulose H4434 (Stem Cell Technologies) and transferred using a blunt needle onto 35 mm dishes (Stem Cell Technologies) in triplicate and incubated at 37° C. and 5° C.O2 for 14 days. Colony Forming Units (CFUs) were scored based on morphological characteristics.

For neuronal differentiation, cells were differentiated at 70% confluency as a monolayer in neuronal differentiation medium (DMEM/F12, Glutamax 1%, B27-Supplement 1%, N2-Supplement 2%, P/S 1% and noggin 20 ng/ml). After 7 days neuronal structures were visible. For endoderm differentiation (AFP stain), cells were differentiated as a monolayer in endoderm differentiation medium (DMEM, B27(-RA) and 100 ng/ml activin-a) for 7 days, then switched to growth medium (DMEM, 10% FBS, 1% P/S) and continued differentiation for 7 days. Primary antibodies used in immunostaining were as follows: Anti-β-Tubulin III (Tuj 1) rabbit anti-human (Sigma, St. Louis, Mo.), 1:500; AFP (h-140) rabbit polyclonal IgG, (Santa Cruz Biotechnology, Santa Cruz, Calif.), 1:100 dilution. All secondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 594 and raised in donkey.

For cardiomyocyte differentiation, colonies were digested at 70% confluency using dispase and placed in suspension culture for embryoid body (EB) formation in differentiation medium (DMEM, 15% FBS, 100 uM ascorbic acid). After 11 days, EBs were plated to adherent conditions using gelatin and the same medium. Beating cardiomyocytes were observed 3 days after replating.

For the teratoma assay, 2.5×10⁶ cells were harvested, spun down, and all excess media was removed. In a 20-week old female SCID mouse, the capsule of the right kidney was gently elevated, and one droplet of concentrated cells was inserted under the capsule. At week 6, when adequate tumor size was observed, the tumor was harvested, fixed in 4% PFA, run through an ethanol gradient, and stored in 70% ethanol. Specimens were sectioned and H&E staining. Slides were imaged with a Leica light microscope.

Myogenic Differentiation of RIPS Cells

Validated RiPS cells were plated into wells coated with 0.1% gelatin (Millipore, Billerica, Mass.), and cultured in DMEM+10% FBS for 4 weeks with passaging every 4-6 days using trypsin. The culture media was switched to Opti-MEM+2% FBS, and the cells were transfected with modified RNA encoding either murine MYOD or GFP the following day, and for the following two days. Media was supplemented with B18R, and replaced 4 hours after each transfection. After the third and final transfection, the media was switched to DMEM+3% horse serum, and cultures were incubated for a further 3 days. Cells were then fixed in 4% PFA and immuno-stained as previously described (Shea et al., 2010). The percentage of myogenin-positive nuclei/total nuclei and nuclei/MyHC-positive myotubes was quantified, with a minimum of 500 nuclei counted per condition.

Thus far, the reprogramming of differentiated cells to pluripotency shows great utility as a tool for studying normal cellular development, while also having the potential for generating patient-specific induced pluripotent stem (iPS) cells that can be used to model disease, or to generate clinically useful cell types for autologous therapies aimed at repairing deficits arising from injury, illness, and aging. Induction of pluripotency was originally achieved by Yamanaka and colleagues by enforced expression of four transcription factors, KLF4, c-MYC, OCT4, and SOX2 (KMOS) using retroviral vectors (Takahashi et al., 2007; Takahashi and Yamanaka, 2006).

A formidable obstacle to therapeutic use of iPS cells has been presented by the requirement for viral integration into the genome. The search for ways to induce pluripotency without incurring genetic change has become the focus of intense research effort. Towards this end, attempts to derive iPS cells using excisable lentiviral and transposon vectors, or through repeated application of transient plasmid, episomal, and adenovirus vectors have been made (Chang et al., 2009; Kaji et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008; Woltjen et al., 2009; Yu et al., 2009). Human iPS cells have also been derived using two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim et al., 2009; Zhou et al., 2009), and transgene delivery using the Sendai virus, which has a completely RNA-based reproductive cycle (Fusaki et al., 2009).

Considerable limitations accompany the non-integrative iPS derivation strategies devised thus far. For example, DNA transfection-based methodologies still entail risk of genomic recombination or insertional mutagenesis, even though they are supposedly safer than viral-based delivery methods. In the protein-based strategies thus far derived, the recombinant proteins used are difficult and challenging to generate and purify in the quantities required, and result in even lower efficiencies of pluripotent stem cell generation that conventional viral-based methods (Zhou et al., 2009). Use of Sendai virus requires stringent steps to purge reprogrammed cells of replicating virus, and the sensitivity of the viral RNA replicase to transgene sequence content can further limit the generality of this reprogramming vehicle (Fusaki et al., 2009). Importantly, the methods discussed that rely on repeat administration of transient vectors, whether DNA or protein-based, have shown very low reprogramming and iPS derivation efficiencies (Jia et al., 2010; Kim et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008; Yu et al., 2009; Zhou et al., 2009), presumably due, without wishing to be bound or limited by theory, to weak or inconstant expression of reprogramming factors.

As demonstrated herein, the inventors have discovered and shown that repeated administration of synthetic, modified messenger RNAs that incorporate novel modifications designed to bypass innate cellular anti-viral responses can reprogram differentiated human cells to pluripotency with conversion efficiencies and kinetics vastly and unexpectedly superior to established protein- and viral-based protocols. Accordingly, described herein are methods and compositions demonstrating that this non-mutagenic, efficient, and highly controllable technology is applicable to a wide range of cellular engineering tasks involving altering cellular developmental potentials, such as the reprogramming of differentiated cells, and the differentiation of reprogrammed cells to a differentiated cell type, such as RNA-iPS (RIPS)-derived fibroblasts to terminally differentiated myogenic cells.

Development of Synthetic, Modified RNAs for Directing Cell Fate

mRNA was manufactured using in vitro transcription (IVT) reactions templated by PCR amplicons (FIG. 1). To promote efficient translation and boost RNA half-life in the cytoplasm, a 5′ guanine cap was incorporated by inclusion of a synthetic cap analog in the IVT reactions (Yisraeli et al., 1989). Within the IVT templates described herein, the open reading frame (ORF) of the gene of interest is flanked by a 5′ untranslated region (UTR) containing a strong Kozak translational initiation signal, and an alpha-globin 3′ UTR terminating with an oligo(dT) sequence for templated addition of a polyA tail.

Cytosolic delivery of mRNA into mammalian cells can be achieved using electroporation or by complexing the RNA with a cationic vehicle to facilitate uptake by endocytosis (Audouy and Hoekstra, 2001; Elango et al., 2005; Holtkamp et al., 2006; Van den Bosch et al., 2006; Van Tendeloo et al., 2001). The latter approach was utilized by the inventors as it would allow for repeated transfection to sustain ectopic protein expression over the days to weeks required for cellular reprogramming. In experiments in which synthetic RNA encoding GFP was transfected into murine embryonic fibroblasts and human epidermal keratinocytes, high, dose-dependent cytotoxicity was noted, which was not attributable to the cationic vehicle, and which was exacerbated on repeated transfections. These experiments demonstrated a serious impediment to achieving sustained protein expression by repeated mRNA transfection.

It is has been reported that exogenous single-stranded RNA (ssRNA) activates antiviral defenses in mammalian cells through interferon and NF-κB dependent pathways (Diebold et al., 2004; Hornung et al., 2006; Kawai and Akira, 2007; Pichlmair et al., 2006; Uematsu and Akira, 2007). In order to increase the sustainability of RNA-mediated protein expression, approaches were sought to reduce the immunogenic profile of the synthetic RNA. The co-transcriptional capping technique yields a significant fraction of uncapped IVT product bearing 5′ triphosphates, which has been reported to trigger the ssRNA sensor RIG-I (Hornung et al., 2006; Pichlmair et al., 2006), and have also been reported to activate PKR, a global repressor of cellular protein translation (Nallagatla and Bevilacqua, 2008). However, treatment of the synthesized RNA with a phosphatase only resulted in modest reductions in the observed cytotoxicity upon repeated transfections.

Eukaryotic mRNA is extensively modified in vivo, and the presence of modified nucleobases has been shown to reduce signaling by RIG-I and PKR, as well as by the less widely expressed but inducible endosomal ssRNA sensors TLR7 and TLR8 (Kariko et al., 2005; Kariko et al., 2008; Kariko and Weissman, 2007; Nallagatla and Bevilacqua, 2008; Nallagatla et al., 2008; Uzri and Gehrke, 2009). In an attempt to further reduce innate immune responses to transfected RNA, mRNAs were synthesized incorporating modified ribonucleoside bases. Complete substitution of either 5-methylcytidine (5mC) for cytidine or pseudouridine (psi) for uridine in GFP-encoding transcripts markedly improved viability and increased ectopic protein expression.

However, the most significant improvements in viability and protein expression were observed when both 5-methylcytidine and pseudouridine were used together (FIGS. 2A-2E). It was discovered that these modifications dramatically attenuated interferon signaling as revealed by qRT-PCR for a panel of interferon response genes, although residual upregulation of some interferon targets was still detected (FIGS. 2F-2K). Innate cellular anti-viral defenses can self-prime through a positive-feedback loop involving autocrine and paracrine signaling by Type I interferons (Randall and Goodbourn, 2008). It was found that media supplementation with a recombinant version of B18R protein, a Vaccinia virus decoy receptor for Type I interferons (Symons et al., 1995), further increased cellular viability following RNA transfection, especially in some cell types. It was discovered that synthesis of RNA with a combination of both modified 5-methylcytidine and pseudouridine ribonucleotides and phosphatase treatment (herein termed “synthetic, modified RNAs”), combined with media supplementation with the interferon inhibitor B18R allowed high, dose-dependent levels of protein expression (FIG. 2L).

It was discovered that transfection of synthetic, modified RNA encoding GFP into six different human cell types resulted in highly penetrant expression (50-90% positive cells), and demonstrated the applicability of these novel methods and compositions to diverse cell types (FIG. 3A). Simultaneous delivery of synthetic, modified RNAs encoding cytosolic-localized red, and nuclear-localized green fluorescent proteins into keratinocytes revealed that generalized co-expression of multiple proteins could be achieved in mammalian cells, and that the resulting proteins were correctly localized to the cytosol and nucleus, respectively (FIG. 2N).

Ectopic protein expression after RNA transfection is transient owing to RNA and protein degradation and the diluting effect of cell division. To establish the kinetics and persistence of protein expression, synthetic, modified RNA encoding GFP variants designed for high and low protein stability (Li et al., 1998) were synthesized and transfected into keratinocytes. Time-course analysis by flow cytometry showed that protein expression persisted for several days for the high-stability variant, but peaked within 12 hours and decayed rapidly thereafter for the destabilized GFP (FIGS. 3B and 3D). These results indicated that a repetitive transfection regimen would be necessary in order to sustain high levels of ectopic expression for short-lived proteins over an extended time course.

To assess this and further address the impact of repeated RNA transfection on cell growth and viability, BJ fibroblasts were transfected daily for 10 days with either unmodified, or synthetic, modified RNAs encoding GFP. It was discovered that daily transfection with synthetic, modified RNA permitted sustained protein expression without substantially compromising the viability of the culture beyond a modest reduction in growth kinetics that was attributable to the transfection reagent vehicle (FIGS. 2O and 3C). Microarray analysis established that prolonged daily transfection with synthetic, modified RNA did not significantly alter the molecular profile of the transfected cells (FIG. 3E), although a modest upregulation of a number of interferon response genes was noted, consistent with the fact that the modifications described herein did not completely abrogate interferon signaling (FIGS. 2F-2K, FIG. 3F). In complete contrast, repeated transfections with unmodified RNA severely compromised the growth and viability of the culture through, in part, elicitation of a massive interferon response (FIGS. 2F-2K), demonstrating that the use of unmodified RNA is not a viable strategy for sustaining long-term polypeptide expression in cells (FIG. 2O).

To determine if modified RNAs could be used to directly alter cell fate, synthetic, modified RNA was synthesized encoding the myogenic transcription factor MYOD (Davis et al., 1987) and transfected into murine C3H10T1/2 cells over the course of 3 days, followed by continued culturing in a low serum media for an additional 3 days. The emergence of large, multi-nucleated myotubes that stained positive for the myogenic markers myogenin and myosin heavy chain (MyHC) provided proof that transfection with synthetic, modified RNAs could be utilized to efficiently direct cell fate (FIG. 2P).

Generation of Induced Pluripotent Stem Cells Using Modified RNAs

The determination of whether induced pluripotent stem cells (iPS) could be derived using synthetic, modified RNAs was next attempted. To this end, synthetic, modified RNAs encoding the four canonical Yamanaka factors, KLF4 (K), c-MYC (M), OCT4 (O), and SOX2 (S), were synthesized, transfected into cells. It was discovered that the synthetic, modified RNAs encoding transcription factors yielded robust protein expression that localized to the nucleus (FIG. 4A). Time-course analysis monitored by flow cytometry yielded expression kinetics and stability similar to destabilized GFP (FIGS. 3B and 3D), demonstrating rapid turnover of these transcription factors (FIGS. 4B-4D). From this, it was concluded that daily transfections would be required to maintain sufficient expression of the Yamanaka factors during long-term, multi-factor reprogramming regimens.

A protocol to ensure sustained high-level protein expression with daily transfection was next discovered by exploring a matrix of conditions encompassing a variety of different transfection reagents, culture media, feeder cell types, and RNA doses. Long-term reprogramming experiments were initiated with human ES-derived dH1f fibroblasts, which display relatively efficient viral-mediated iPS cell conversion (Chan et al., 2009; Park et al., 2008). Low-oxygen (5% O2) culture conditions and a KMOS stoichiometry of 1:1:3:1 were also employed, as these have been reported to promote efficient iPS conversion in viral-based methods (Kawamura et al., 2009; Papapetrou et al., 2009; Utikal et al., 2009; Yoshida et al., 2009). Synthetic, modified RNA encoding a short half-life nuclear GFP was spiked into the KMOS RNA cocktail to allow visualization of continued protein expression from modified RNA during the course of the experiment (FIGS. 5A-5B). Experiments conducted in this manner revealed widespread transformation of fibroblast morphology to a compact, epithelioid morphology within the first week of synthetic, modified RNA transfection, which was followed by emergence of canonical hES-like colonies with tight morphology, well-defined borders, and prominent nucleoli (FIG. 5C). RNA transfection was terminated on day 17, and three days later colonies were mechanically picked and expanded to establish 14 prospective iPS lines, designated dH1f-RiPS (RNA-derived iPS) 1-14.

It was next attempted to reprogram somatically-derived cells to pluripotency using a similar reprogramming regimen. A five-factor cocktail including a modified RNA encoding LIN28 (KMOSL) (Yu et al., 2007) was employed and the media was supplemented with valproic acid (VPA), a histone deacetylase inhibitor, which has been reported to increase reprogramming efficiency (Huangfu et al., 2008). Four human cell types were tested: Detroit 551 (D551) and MRC-5 fetal fibroblasts, BJ post-natal fibroblasts, and fibroblast-like cells cultured from a primary skin biopsy taken from an adult cystic fibrosis patient (CF cells). Daily transfection with the modified RNA KMOSL cocktail gave rise to numerous hES-like colonies in the D551, BJ, and CF cultures that were mechanically picked at day 18, while MRC-5-derived colonies were picked at day 25. Multiple RiPS colonies were expanded for each of the somatic lines, and immunostaining confirmed the expression of hES markers TRA-1-60, TRA-1-81, SSEA3, SSEA4, OCT4, and NANOG in all the RiPS lines examined (FIG. 4F, FIG. 5C). Three RiPS cell clones from each of these four derivations were analyzed and confirmed to originate from the seeded somatic cells by DNA fingerprinting, and all presented normal karyotypes. In the experiments described above, the transfected fibroblast cultures were passaged once at an early time point (day 6 or 7) in order to promote fibroblast proliferation, which has been shown to facilitate reprogramming (Hanna et al., 2009). However, in independent experiments, RiPS cells were also derived from BJ and Detroit 551 fibroblasts in the absence of cell passaging, indicating that this was not required for modified RNA iPS-derivation (FIGS. 6A-6B).

Molecular Characterization and Functional Potential of RiPS Cells

A number of molecular and functional assays were performed to assess whether the RiPS cells described herein had been reprogrammed to pluripotency (Table 6). Multiple RiPS lines derived from each of the five starting cell types were evaluated by quantitative RT-PCR (qRT-PCR), and all demonstrated robust expression of the pluripotency-associated transcripts OCT4, SOX2, NANOG, and hTERT (FIG. 7A). RiPS clones derived from dH1f, MRC5, BJ, and CF fibroblasts were further analyzed by bisulfite sequencing, which revealed extensive demethylation of the OCT4 locus relative to the parental fibroblasts, an epigenetic state equivalent to human ES cells (FIG. 7B).

TABLE 6 Pluripotency validation assays performed in this study. Bisulfite Developmental Potential Immunostaining# qRT-PCR Sequencing^(Ω) Microarray In vitro Teratoma dH1F-RiPS-1.3 dH1F-RiPS-1.2 dH1F-RiPS-1.2 dH1F-RiPS-1.2 dH1F-RiPS-1.2{circumflex over ( )}^(†ø)* dH1F-RiPS-1.3 dH1F-RiPS-1.6 dH1F-RiPS-1.3 dH1F-RiPS-1.3 dH1F-RiPS-1.3 dH1F-RiPS-1.6{circumflex over ( )}^(ø) dH1F-RiPS-1.5 dH1F-RiPS-1.13 dH1F-RiPS-1.6 dH1F-RiPS-1.6 dH1F-RiPS-1.6 dH1F-RiPS-1.13{circumflex over ( )}^(ø) dH1F-RiPS-1.6 BJ-RiPS-1.1 dH1F-RiPS-1.7 BJ-RiPS-1.2 dH1F-RiPS-1.7 dH1F-RiPS-1.14{circumflex over ( )}^(ø) dH1F-RiPS-1.7 BJ-RIPS-1.2 BJ-RiPS-1.1 BJ-RIPS-1.3 BJ-RiPS-1.1 MCR5-RiPS-1.8{circumflex over ( )}^(†)* dH1F-RiPS-1.11 BJ-RiPS-1.3 BJ-RiPS-1.2 MCR5-RiPS-1.8 BJ-RiPS-1.2 MCR5-RiPS-1.9{circumflex over ( )}^(†)* BJ-RiPS-1.1 MCR5-RiPS-1.2 BJ-RiPS-1.3 MCR5-RiPS-1.9 BJ-RiPS-1.3 MCR5-RiPS- BJ-RiPS-1.2 MCR5-RiPS-1.3 MCR5-RiPS-1.8 MCR5-RiPS-1.11 MCR5-RiPS-1.8 BJ-RiPS-1.1{circumflex over ( )}^(†ø)* CF-RiPS-1.2 MCR5-RiPS-1.4 MCR5-RiPS-1.9 CF-RiPS-1.2 MCR5-RiPS-1.9 BJ-RiPS-1.2{circumflex over ( )}^(†ø)* CF-RiPS-1.2 MCR5-RiPS-1.11 CF-RiPS-1.3 MCR5-RiPS-1.11 BJ-RiPS-1.3{circumflex over ( )}^(†)* CF-RiPS-1.3 CF-RiPS-1.2 CF-RiPS-1.4 CF-RiPS-1.2 CF-RiPS-1.2{circumflex over ( )}^(†)* CF-RiPS-1.4 CF-RiPS-1.3 CF-RiPS-1.3 CF-RiPS-1.3{circumflex over ( )}^(†)* D551-RiPS-1.1 CF-RiPS-1.4 CF-RiPS-1.4 CF-RiPS-1.4{circumflex over ( )}^(ø)* D551-RiPS-1.2 D551-RiPS-1.1 D551-RiPS-1.1{circumflex over ( )}^(†)* D551-RiPS-1.3 D551-RiPS-1.2 D551-RiPS-1.2{circumflex over ( )}* D551-RiPS-1.3 D551-Ri1PS-1.3{circumflex over ( )}* Table 6 shows the RiPS clones that were validate in each assay. # Validated for immune-staining for all of TRA-1-60, TRA-1-80, SSEA3, SSEA4, OCT4, NANOG. Ω Demethylation of the OCT4 promoter. In vitro differentiation including ^embryoid body formation, øtrilineage by directed differentiation, † beating cardiomyocytes, and * blood formation by CFC assays in methylcellulose.

To gain more global insight into the molecular properties of RiPS cells, gene expression profiles of RiPS clones from multiple independent derivations were generated and compared to fibroblasts, human embryonic stem (ES) cells, and virally-derived iPS cell lines. These analyses revealed that all synthetic, modified RNA-derived iPS clones examined had a molecular signature that very closely recapitulated that of human ES cells while being highly divergent from the profile of the parental fibroblasts (FIGS. 7C-7H). Importantly, pluripotency-associated transcripts including SOX2, REX1, NANOG, OCT4, LIN28 and DNMT3B were substantially upregulated in the RiPS cells compared to the parental fibroblast lines to levels comparable to human ES cells (FIGS. 7C-7H). Furthermore, when the transcriptional profiles were subjected to unsupervised hierarchical clustering analysis, all RiPS clones analyzed clustered more closely to human ES cells than did virally-derived iPS cells, indicating that synthetic, modified RNA-derived iPS cells more fully recapitulated the molecular signature of human ES cells (FIG. 7I).

To evaluate the developmental potential of RiPS cells, embryoid bodies (EBs) were generated from multiple clones representing five independent RiPS derivations. Beating cardiomyocytes were observed for vast majority of the EBs (Table 6). Mesodermal potential was further evaluated in methylcellulose assays which showed that all lines tested were able to differentiate into hematopoietic precursors capable of giving rise to colony numbers and a spectrum of blood colony types comparable to human ES cells (FIG. 8A, Table 6). A subset of clones was further plated onto matrigel and differentiated into Tuj1-positive neurons (ectoderm), and alpha-fetoprotein-positive endodermal cells (FIG. 8B, Table 6). Finally, tri-lineage differentiation potential was confirmed in vivo by the formation of teratomas from dH1F-, CF- and BJ-RiPS cells, that histologically revealed cell types of the three germ layers (FIG. 8C, FIG. 9, Table 6).

Taken together, these data demonstrate by the most stringent molecular and functional criteria available in regard to human pluripotent cells (Chan et al., 2009; Smith et al., 2009), that the synthetic, modified RNA-derived iPS clones from multiple independent derivations described herein were reprogrammed to pluripotency, and closely recapitulated the functional and molecular properties of human ES cells. Significantly, these synthetic, modified RNA-derived iPS clones had molecular properties more similar to human ES cells than did cells that were reprogrammed using standard, viral-based methods.

Modified RNAs Generate iPS Cells at Very High Efficiency

During the course of the experiments, surprisingly high reprogramming efficiencies and rapid kinetics of iPS cell generation using the synthetic, modified RNAs described herein were observed. To quantify the efficiency of RiPS derivation more thoroughly, a number of reprogramming experiments were undertaken and results quantitated based on the expression of the iPS-specific markers TRA-1-60 and TRA-1-81, (Chan et al., 2009; Lowry et al., 2008). In one set of experiments, BJ fibroblasts transfected with a five-factor modified RNA cocktail (KMOSL), this time without the use of VPA, demonstrated an iPS conversion efficiency of over 2%, which is two orders of magnitude higher than typically reported for virus-based derivations (FIGS. 10A-10B, Table 7). Moreover, in contrast to virus-mediated BJ-iPS derivations, in which iPS colonies typically take around 4 weeks to emerge, by day 17 of RNA transfection the plates had already become overgrown with ES-like colonies (FIG. 10A).

TABLE 7 Quantification of reprogramming efficiency. Well Efficiency Experiment Cells plated Split Condition fraction Colonies/well (%) BJ (KMOSL) 300,000 d7 Y27632−  1/24 249 ± 21  2.0 Y27632+  1/24 326 ± 49  2.6 4-Factor  50,000 d6 4F 20% O₂ 1/6 48 ± 18 0.6 (KMOS) vs. 4F 5% O₂ 1/6 228 ± 30  2.7 5-Factor 5F 20% O₂ 1/6 243 ± 42  2.9 (KMOSL) 5F 5% O₂ 1/6 367 ± 38  4.4 RNA vs. Virus 100,000 d6 Virus 1/3  13 ± 3.5 0.04 (KMOS) RNA 1/6 229 ± 39  1.4 For each experimental condition, efficiency was calculated by dividing the average count of TRA-1-60-positive colonies per well by the initial number of cells plated, scaled to the fraction of cells replated in each well. Cultures were passaged at day 6 or 7 as indicated. The BJ experiment was started in a 10-cm dish, dH1f trials in individual wells of a 6-well plate. Colony counts are shown ±s.d., n=6, except in the RNA vs. Virus trial, where n=9 for virus, n=18 for RNA.

In another set of experiments, the contributions of low-oxygen culture and LIN28 to the efficiency of RiPS derivation were evaluated. The yield of TRA-1-60/TRA-1-81-positive colonies in the ambient (20%) oxygen condition was four-fold lower than in the cultures maintained at 5% O2 when using KMOS RNA, but this deficit was negated when LIN28 was added to the cocktail (FIGS. 10C-10D, Table 7). The highest conversion efficiency (4.4%), which is higher than any reported conversion efficiency, was observed when low-oxygen culture and the five-factor KMOSL cocktail were combined.

To directly compare the kinetics and efficiency of the RiPS derivation protocol against an established viral protocol, an experiment in which dH1f fibroblasts were transfected with KMOS synthetic, modified RNAs, or transduced with KMOS retroviruses in parallel was conducted. As had been observed in the previous experiments described herein, ES-like colonies began to emerge by day 13 from the synthetic, modified RNA-transfected cultures, and the plates became overgrown with ES-like colonies by the 16th and final day of transfection. These synthetic, modified RNA-derived cultures were therefore fixed for analysis on day 18 (FIGS. 10E-10G). Notably, at this time, no ES-like colonies had appeared in the retrovirally transduced cultures, and colonies only began to emerge on the 24th day post-transduction, which is a time point consistent with previous reports describing iPS derivations by retroviruses (Lowry et al., 2008; Takahashi et al., 2007). These retroviral-derived cultures were fixed for analysis on day 32. Both arms of the experiment were then immunostained and TRA-1-60-positive colonies were counted. These experiments revealed that the kinetics of modified RNA iPS derivation were almost twice as fast as retroviral iPS derivation. Further, and importantly, iPS derivation efficiencies were 1.4% for synthetic, modified RNA cultures, and only 0.04% for retroviral cultures, corresponding to a surprising 36-fold higher conversion efficiency with the synthetic, modified RNA compositions and protocols (FIGS. 10E-10G, Table 7). Thus, by the combined criteria of colony numbers and kinetics of reprogramming, the efficiency of synthetic, modified RNA iPS derivation greatly exceeds that of conventional retroviral approaches.

Utilization of Synthetic, Modified RNA to Direct Differentiation of Pluripotent RiPS Cells to a Terminally-Differentiated Cell Fate.

To realize the promise of iPS cell technology for regenerative medicine or disease modeling, it is imperative that the multi-lineage differentiation potential of pluripotent cells be harnessed. Although limited progress has been made in directing the differentiation of pluripotent ES cells to various lineages by modulating the extracellular cytokine milieu, such protocols remain inefficient. Given the high efficiency of iPS derivation by the novel synthetic, modified RNAs and methods thereof described herein, whether this technology could also be utilized to redirect pluripotent or multipotent cells towards differentiated cell fates was also determined. To test this, one of the validated RiPS lines described herein was subjected to an in vitro differentiation protocol in which FGF was withdrawn, serum added, and the cells plated onto gelatin (FIGS. 11A-11C). Cells obtained under these conditions were subjected to three consecutive days of transfection with a MYOD-encoding synthetic, modified RNA to provoke myogenic differentiation. The cells were then cultured an additional three days and then immunostained for the myogenic markers myogenin and MyHC, which revealed a high percentage of large multi-nucleated myogenin and MyHC double positive myotubes (FIGS. 11A-11C).

Taken together, the experiments described herein provide clear proof that synthetic, modified RNAs can be used to both reprogram cells to a pluripotent state at high and unexpected efficiencies, and also direct the fate of such cells and other pluripotent or multipotent cells to cells having lower developmental potential, such as a terminally differentiated somatic cell type.

Discussion

Described herein are novel compositions and technologies that use a combination of synthetic RNA modifications, and in some embodiments, a soluble interferon inhibitor, to overcome innate anti-viral responses and permit repeated transfections with RNA, thus enabling highly efficient alterations in cellular phenotypes and developmental potentials, such as highly efficient reprogramming of somatic cells to pluripotency, and directing the differentiation of pluripotent cells towards a desired lineage. The novel methodologies and compositions described herein offer several key advantages over established reprogramming techniques. By obviating the need to perform experiments under stringent biological containment, synthetic, modified RNA technology makes reprogramming accessible to a wider community of researchers. More fundamentally, the approaches described herein allow protein stoichiometry to be regulated globally within cultures, while avoiding the stochastic variation of expression typical of integrating vectors, as well as the uncontrollable and undesired effects of viral silencing. Given the stepwise character of the phenotypic changes observed during pluripotency induction (Chan et al., 2009; Smith et al., 2010), individual transcription factors can play distinct, stage-specific roles during reprogramming. The unprecedented potential for temporal control over factor expression afforded by the technologies described herein can help researchers unravel these nuances, yielding further insights that can be applied to further enhance the efficiency and kinetics of reprogramming.

While the risk of mutagenesis is a major safety concern holding back clinical exploitation of induced pluripotency, other factors also play a role. It has become increasingly apparent that all iPS cells are not created equal with respect to epigenetic landscape and developmental plasticity (Hu et al., 2010; Miura et al., 2009). In this regard, the most stringent molecular and functional criteria for reprogramming human cells have been applied herein (Chan et al., 2009; Smith et al., 2009), to demonstrate that the iPS clones derived from synthetic, modified RNAs from multiple independent derivations were reprogrammed to pluripotency, and also closely recapitulated the functional and molecular properties of human ES cells. Significantly, as described herein, synthetic, modified RNA derived iPS cells more faithfully recapitulated the global transcriptional signature of human ES cells than retrovirally-derived iPS cells, indicating that the compositions and methods for RNA reprogramming described herein produce higher quality iPS cells, possibly owing, without wishing to be bound or limited by theory, to the fact that they are transgene-free.

The transient and non-mutagenic character of RNA-based protein expression can also deliver important clinical benefits, in some embodiments, outside the domain of lineage reprogramming and alteration of cellular developmental potential. The use of RNA transfection to express cancer or pathogen antigens for immunotherapy is already an active research area (Rabinovich et al., 2008; Rabinovich et al., 2006; Van den Bosch et al., 2006; Weissman et al., 2000), and the synthetic, modified RNA can be used, in some embodiments, to transiently express surface proteins, such as homing receptors, to target cellular therapies toward specific organs, tissues, or diseased cells (Ryser et al., 2008).

For tissue engineering to progress further, there is a pressing need for safe and efficient means to alter cellular fates. In terms of personalized medicine applications, iPS cells are a starting point for patient-specific therapies, and specification of clinically useful cell types is required to produce autologous tissues for transplantation or for disease modeling. Importantly, the inventors have demonstrated that the synthetic, modified RNA-based technologies described herein that enable highly efficient reprogramming, can are equally applicable to efficiently alter pluripotent cell fate to terminally differentiated fates without compromising genomic integrity. In light of these considerations, the novel compositions and approaches described herein can become central enabling technology for cell-based therapies and regenerative medicine. 

We claim:
 1. A method of making induced pluripotent stem cells, the method comprising contacting mammalian somatic cells in vitro with synthetic, modified RNAs encoding OCT4, SOX2, and KLF4 at least three times, wherein each cytosine of each of the synthetic modified RNAs is replaced with 5-methylcytosine and each uracil of each of the synthetic modified RNAs is replaced with pseudouracil, such that iPS cells are obtained.
 2. The method of claim 1, wherein each of the synthetic, modified RNAs encoding OCT4, SOX2, and KLF4 further comprises a 5′ cap.
 3. The method of claim 2, wherein the 5′ cap is a 5′ cap analog.
 4. The method of claim 3, wherein the 5′ cap analog is a 5′ diguanosine cap.
 5. The method of claim 1, wherein each of the synthetic, modified RNAs encoding OCT4, SOX2, and KLF4 does not comprise a 5′ triphosphate.
 6. The method of claim 1, wherein each of the synthetic, modified RNAs encoding OCT4, SOX2, and KLF4 further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof. 